Bioconjugation technical handbook Reagents for crosslinking, immobilization, modification, biotinylation, and fluorescent labeling of proteins and peptides Introduction

Bioconjugation is the process of chemically joining two or more molecules or biomolecules by a covalent bond. This technique utilizes a variety of reagents for the crosslinking, immobilization, modification, and labeling of proteins and peptides. Bioconjugation reagents contain reactive ends to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. The availability of several chemical groups in proteins and peptides make them targets for a wide range of applications, including biotinylation, immobilization to solid supports, protein structural studies, and metabolic labeling. Chemical agents may be used to modify amino acid side chains on proteins and peptides in order to alter charges, block or expose reactive binding sites, inactivate functions, or change functional groups to create targets for crosslinking and labeling. Crosslinking, labeling, and modification reagents can be described by their chemical reactivity, molecular properties, or applications (Table 1). Table 1. Key considerations for selecting the right bioconjugation reagent. Chemical reactivity First, select a reagent with the functional group(s) to bind your biomolecules of interest. NHS ester reaction Maleimide reaction

pH 6.5–7.5

Amine-containing NHS ester Amine bond NHS Sulfhydryl- Maleimide Thioether bond molecule compound containing compound molecule

Molecular properties Second, choose which features or characteristics are important for your application. Functionality Cleavability Structural modifications

OH

O O O O O O N HO P N O O O O O

DSS O OH NH2 HO O Disuccinimidyl suberate CA(PEG)TCEP n n MW 368.34 M.W. 250.15 DSS—same reactivity DSP with disulfide linker for cleavage TCEP—reducesCarboxy-PEG disulfiden-amine bonds Spacer arm 11.4 Å on both ends Carboxyl-(ethyleneglycol)n ethylamine n = 8 CA(PEG)8 Spacer arm composition Spacer arm length Spacer arm structure MW 441.51 18.1Å Spacer Arm 33.6Å O O O O O O n = 12 N O O CA(PEG)12 N N N HO O O NH 2 MW 617.72 O O O O Spacer Arm 46.8Å O CA(PEG)4 BMH AMAS MW 265.30 n = 24 M.W. 276.29 BMH with hydrocarbon spacer AMAS 4.4 Å—shortM.W. 252.18 spacer between CA(PEG)Spacern—adds Arm 18.1Å solubility in CA(PEG)24 Spacer Arm 16.1 Å reactiveSpacer Arm groups 4.4 Å aqueous solutions MW 1146.35 Applications Spacer Arm 89.8Å Select the specific reagent depending on the application (e.g., protein detection, immobilization, or interaction studies). Labeling Immobilization Protein interaction studies

O O NH O O O HN O O O S O O N N N N O HO S O O O O O O O O DSS DSSO Disuccinimidyl sulfoxide Biotin—for labeling DSS—couplesDisuccinimidyl proteins sube ratote surfaces DSSO—MS-cleavable crosslinker MW 388.35 MW 368.34 and detection Spacer Arm 10.3 Å Spacer arm 11.4 Å Packaging options Select a package size or grade based on the scale of your reaction or your requirements. These reagents are available from milligram to kilogram quantities. Milligram Milligram to gram Milligram to gram Gram to kilogram

Bulk-size packages available

Single- tubes Catalog product Premium grade Large-volume or custom packages Contents

Chemical reactivity of Hapten–carrier conjugation for bioconjugation reagents antibody production 27 Introduction 5 Protein–protein conjugation 28 Amine-reactive chemical groups 6 Creation of immunotoxins 29 Carboxylic acid–reactive Label transfer 29 chemical groups 7 Subunit crosslinking and protein Sulfhydryl-reactive chemical groups 8 structural studies 31 Carbonyl-reactive chemical groups 10 Protein interaction and crosslinking Nonspecificly-reactive chemical groups 12 using mass spectrometry 32 Chemoselective ligation 13 MS-cleavable crosslinkers 33 In vivo crosslinking 34 Molecular properties of Metabolic labeling 35 bioconjugation reagents Cell surface crosslinking 36 Introduction 14 Cell membrane structural studies 36 Homobifunctional and heterobifunctional crosslinkers 15 Special packaging to meet specific General reaction conditions 16 bioconjugation needs Modifications 16 Introduction 37 Spacer arm length 18 No-Weigh packaging format for Spacer arm composition 18 bioconjugation reagents 38 Spacer arm cleavability 18 Premium-grade bioconjugation reagents 39 Spacer arm structure and solubility 19 Bioconjugation resources 40 Applications using bioconjugation reagents Glossary 42 Introduction 20 Protein and peptide biotinylation 21 References 44 Fluorescent labeling of proteins and peptides 23 Ordering information 50 Protein immobilization Related handbooks and resources 71 onto solid supports 25 Surface modification using PEG-based reagents 26 Chemical reactivity of biconjugation reagents

Introduction The most important property of a bioconjugation reagent is its reactive chemical group. The reactive group establishes the method and mechanism for chemical modification. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids. Protein modification reagents like PEGylation or biotinylation reagents have a reactive group at one terminus (PEG chain or biotin group, respectively) and a chemical moiety at the other end. The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids (Figure 1, Table 1). Coupling can also be nonselective using photoreactive groups.

5 O Amine-reactive chemical groups

H2N O OH O H N H N 2 OH 2 OH Primary amines (–NH2) exist at the N-terminus of each R polypeptide chain (called the α-amine) and in the side chain HS of lysine (Lys, K) residues (called the ε-amine). Because of NH2 their positive charge at physiological conditions, primary Generic Lysine Cysteine amino acid amines are usually outward facing (i.e., on the outer surface of proteins), making them more accessible for conjugation without denaturing protein structure. A number of reactive O O O O O O O H H H H H H H3N N N N N N N chemical groups target primary amines (Figure 2), but the C C C C C C O R H H HO OH H most commonly used groups are N-hydroxysuccinimide SH esters (NHS esters) and imidoesters. O O HO O R N CS O O Isothiocyanate Cl R Gly Glu Gly Asp Gly Cys R S Cl R N NH C R N CO O H N Figure 1. Common amino acid functional groups targeted Isocyanate Sulfonyl chloride Aldehyde Carbodiimide O for bioconjugation. O O F O R Rʹ O O R N R N R O Carbonate Acyl azide Anhydride Fluorobenzene F O F F Table 2. Popular crosslinker reactive groups for protein conjugation. O Target N NH2 O HO R R O F R CH3 functional O O F Reactivity class group Reactive chemical group NHS ester Imidoester Epoxide Fluorophenyl ester

Amine-reactive –NH2 NHS ester Imidoester Figure 2. Reactive chemical groups that target primary amines. Pentafluorophenyl ester Hydroxymethyl phosphine Carboxyl-to-amine reactive –COOH Carbodiimide (e.g., EDC) Sulfhydryl-reactive –SH Maleimide N-hydroxysuccinimide esters (NHS esters) Haloacetyl (bromo-, chloro-, or iodo-) Pyridyl disulfide O NHS esters are reactive groups formed by O Thiosulfonate N EDC activation of carboxylate molecules. O Vinyl sulfone R O NHS ester–activated crosslinkers and Aldehyde-reactive –CHO Hydrazide (e.g., oxidized Alkoxyamine NHS ester labeling compounds react with primary sugars, carbonyls) NHS ester amines in slightly alkaline conditions to Photoreactive Random Diazirine (i.e., nonselective, Aryl azide yield stable amide bonds (Figure 3). The reaction releases random insertion) N-hydroxysuccinimide, which can be removed easily by Hydroxyl (nonaqueous)- –OH Isocyanate dialysis or desalting. reactive

Azide-reactive –N3 Alkyne Phosphine NHS-reactive chemistry NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate–bicarbonate, HEPES, or borate buffers at pH 7.2–8.5 for 30 minutes to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) are not compatible, because they compete for the reaction. However, in some procedures it is useful to add Tris or glycine buffer at the end of a conjugation procedure to stop the reaction. Learn more at thermofisher.com/proteincrosslinking

6 O O O O Imidoester reaction chemistry P N NH N O 2 pH 7–9 N R + P R H + HO Imidoester crosslinkers react rapidly with amines at O O alkaline pH to form amidine bonds but have short half- NHS ester Primary amine Stable conjugate NHS reagent on protein (amide bond) lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases, making crosslinking more Figure 3. NHS ester reaction scheme for chemical conjugation efficient when performed at pH 10 than at pH 8. Reaction to a primary amine. (R) represents a labeling reagent or one end of a crosslinker having the NHS ester reactive group; (P) represents a conditions below pH 10 may result in side reactions, protein or other molecule that contains the target functional group although amidine formation is favored between pH 8 and (i.e., primary amine). 10. Studies using monofunctional alkyl imidates reveal that at pH <10, conjugation can form with just one imidoester Hydrolysis of the NHS ester competes with the primary functional group. An intermediate N-alkyl imidate forms amine reaction. The rate of hydrolysis increases with buffer at the lower pH range and will either crosslink to another pH and contributes to less-efficient crosslinking in less- amine in the immediate vicinity, resulting in N,N´-amidine concentrated protein solutions. The half-life of hydrolysis derivatives, or it will convert to an amidine bond. At higher for NHS ester compounds is 4–5 hours at pH 7.0 and 0°C. pH, the amidine is formed directly without formation of This half-life decreases to 10 minutes at pH 8.6 and 4°C. an intermediate or side product. Extraneous crosslinking The extent of NHS ester hydrolysis in aqueous solutions that occurs below pH 10 sometimes interferes with free of primary amines can be measured at 260–280 nm, interpretation of results when thiol-cleavable diimidoesters because the NHS byproduct absorbs in that range. are used.

NH + NH + 2 2 P Sulfo-NHS esters are identical to NHS esters except NH O 2 pH 8–9 N R ++P R H CH3OH that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This charged group has Imidoester Primary amine Conjugate no effect on the reaction chemistry, but it does tend to reagent on protein (amidine bond) increase the water solubility of crosslinkers containing Figure 4. Imidoester reaction scheme for chemical conjugation them. In addition, the charged group prevents sulfo-NHS to a primary amine. (R) represents a labeling reagent or one end of crosslinkers from permeating cell membranes, enabling a crosslinker having the imidoester reactive group; (P) represents a them to be used for cell surface crosslinking methods. protein or other molecule that contains the target functional group (i.e., primary amine, –NH2). Imidoesters Carboxylic acid–reactive

NH + 2 Imidoester crosslinkers react with primary chemical groups O R amines to form amidine bonds (Figure 4). To ensure specificity for primary amines, Carboxylic acids (–COOH) exist at the C-terminus of each Imidoester imidoester reactions are best done in amine- polypeptide chain and in the side chains of aspartic acid free, alkaline conditions (pH 10), such as with borate buffer. (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxyls are usually on the surface of protein structure. Because the resulting amidine bond is protonated, the Carboxylic acids are reactive towards carbodiimides. crosslink has a positive charge at physiological pH, much like the primary amine that it replaced. For this reason, Carbodiimides (EDC and DCC) imidoester crosslinkers have been used to study protein structure and molecular associations in membranes EDC and other carbodiimides are +Cl− N NH and to immobilize proteins onto solid-phase supports C zero-length crosslinkers. They cause N while preserving the isoelectric point (pI) of the native Carbodiimide (EDC) direct conjugation of carboxylates protein. Although imidoesters are still used in certain (–COOH) to primary amines (–NH2) procedures, the amidine bonds formed are reversible without becoming part of the final amide-bond crosslink at high pH. Therefore, the more stable and efficient between target molecules. NHS ester crosslinkers have steadily replaced them in most applications.

7 Because peptides and proteins contain multiple Carboxylic Primary amine O Stable conjugate acid 2 NH (amide bond) O 2 N carboxyls and amines, direct EDC-mediated crosslinking Cl– 2 1 H OH H usually causes random polymerization of polypeptides. 1 N+ Nevertheless, this reaction chemistry is used widely in O Hydrolysis H O 2 OH O N 1 immobilization procedures (e.g., attaching proteins to a O – O NH O Cl 1 S – carboxylated surface) and in immunogen preparation (e.g., +NH O O O attaching a small peptide to a large carrier protein). O-acylisourea N intermediate O O 1 NH2 N O O (unstable) S O– 2 C O N Amine-reactive EDC reaction chemistry EDC sulfo-NHS ester Primary crosslinker N HO (dry-stable) amine EDC reacts with carboxylic acid groups to form an active O O-acylisourea intermediate that is easily displaced by Sulfo-NHS nucleophilic attack from primary amino groups in the Figure 6. Carboxyl-to-amine crosslinking using the carbodiimide reaction mixture (Figure 5). The primary amine forms EDC and sulfo-NHS. Addition of NHS or sulfo-NHS to EDC reactions an amide bond with the original carboxyl group, and an (bottom-most pathway) increases efficiency and enables molecule (1) to be activated for storage and later use. EDC by-product is released as a soluble urea derivative. The O-acylisourea intermediate is unstable in aqueous EDC is also capable of activating phosphate groups in solutions. Failure to react with an amine results in hydrolysis the presence of imidazole for conjugation to primary of the intermediate, regeneration of the carboxyls, and the amines. The method is sometimes used to modify, release of an N-unsubstituted urea. label, crosslink, or immobilize oligonucleotides through their 5´ phosphate groups. Primary amine − H H Cl N+ N+ H N+ H2N 2 O DCC reaction chemistry and applications O 2 OH O N NH DCC (dicyclohexyl carbodiimide) crosslinks carboxylic + N + 1 N 1 H O NH O NH C 1 acids to primary amines in the same manner as EDC. N However, because DCC is not water-soluble, it is primarily Carboxylic O-acylisourea Crosslinked Isourea acid EDC active ester protein by-product used in manufacturing and organic synthesis applications rather than in the typical protein research biology lab. For Figure 5. Carboxyl-to-amine crosslinking using the popular carbodiimide EDC. Molecules (1) and (2) can be peptides, proteins, or example, most commercially available, ready-to-use NHS any chemicals that have respective carboxylate and primary amine groups. ester crosslinkers and labeling reagents are manufactured When they are peptides or proteins, these molecules are tens to thousands using DCC. Because water is excluded, the resulting NHS of times larger than the crosslinker and conjugation arms diagrammed in the reaction. ester can be prepared and stabilized as a dried powder without appreciable hydrolysis. DCC is also commonly EDC crosslinking is most efficient in acidic (pH 4.5) used in commercial peptide synthesis operations. conditions and must be performed in buffers devoid of extraneous carboxyls and amines. MES buffer Sulfhydryl-reactive chemical groups (4-morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate buffers and neutral pH (up to Sulfhydryls (–SH) exist in the side chain of cysteine (Cys, 7.2) conditions are compatible with the reaction chemistry, C). Often as part of a protein’s secondary or tertiary but with lower efficiency. Increasing the amount of EDC in a structure, cysteines are joined between their side chains reaction solution can compensate for the reduced efficiency. via disulfide bonds (–S–S–). These must be reduced to sulfhydryls to make them available for crosslinking by most N-hydroxysuccinimide (NHS) or its water-soluble analog types of reactive groups. Sulfhydryls are reactive towards (sulfo-NHS) is often included in EDC coupling protocols maleimides, haloacetyls, and pyridyl disulfides. to improve efficiency or create dry-stable (amine-reactive) intermediates (Figure 6). EDC couples NHS to carboxyls, forming an NHS ester that is considerably more stable than the O-acylisourea intermediate while allowing efficient conjugation to primary amines at physiological pH.

8 Maleimides does not contain thiols and does have to be removed before reactions using maleimide reagents. Maleimide-activated crosslinkers and labeling O N reagents react specifically with sulfhydryl Excess maleimides can be quenched at the end of a R O groups (–SH) at near-neutral conditions reaction by adding free thiols. EDTA can be included in Maleimide (pH 6.5–7.5) to form stable thioether linkages. the coupling buffer to chelate stray divalent metals that Disulfide bonds in protein structures must be otherwise promote oxidation of sulfhydryls (nonreactive). reduced to free thiols (sulfhydryls) to react with maleimide reagents. Extraneous thiols (e.g., from most reducing Haloacetyls agents) must be excluded from maleimide reaction buffers because they will compete for coupling sites. Most haloacetyl crosslinkers contain an H N I iodoacetyl or a bromoacetyl group. R Short homobifunctional maleimide crosslinkers enable O Haloacetyls react with sulfhydryl groups Iodoacetyl group disulfide bridges in protein structures to be converted to at physiological to alkaline conditions permanent, irreducible linkages between cysteines. More (pH 7.2–9), resulting in stable thioether commonly, the maleimide chemistry is used in combination linkages. To limit free iodine generation, which has the with amine-reactive NHS ester chemistry in the form of potential to react with tyrosine, histidine, and tryptophan heterobifunctional crosslinkers that enable controlled two- residues, it is best to perform iodoacetyl reactions in step conjugation of purified peptides and/or proteins. the dark.

Maleimide reaction chemistry Haloacetyl reaction chemistry The maleimide group reacts specifically with sulfhydryl Haloacetyls react with sulfhydryl groups at physiological groups when the pH of the reaction mixture is between pH. The reaction of the iodoacetyl group proceeds by pH 6.5 and 7.5, resulting in the formation of a stable nucleophilic substitution of iodine with a sulfur atom from thioether linkage that is not reversible (Figure 7). In a sulfhydryl group, resulting in a stable thioether linkage more alkaline conditions (pH >8.5), the reaction favors (Figure 8). Using a slight excess of the iodoacetyl group primary amines and also increases the rate of hydrolysis over the number of sulfhydryl groups at pH 8.3 ensures of the maleimide group to a nonreactive maleamic sulfhydryl selectivity. In the absence of free sulfhydryls, or if acid. Maleimides do not react with tyrosines, histidines, a large excess of iodoacetyl group is used, the iodoacetyl or methionines. group can react with other amino acids. Imidazoles can react with iodoacetyl groups at pH 6.9–7.0, but the S O O P incubation must proceed for longer than one week. N SH pH 6.5–7.5 N R O + P R O H H P N SH pH >7.5 N Maleimide Sulfhydryl Stable conjugate I S reagent on protein (thioether bond) R ++P R HI O O Iodoacetyl Sulfhydryl Conjugate Figure 7. Maleimide reaction scheme for chemical conjugation to a reagent on protein (thioether bond) sulfhydryl. (R) represents a labeling reagent or one end of a crosslinker having the maleimide reactive group; (P) represents a protein or other Figure 8. Iodoacetyl reaction scheme for chemical conjugation molecule that contains the target functional group (i.e., sulfhydryl, –SH). to a sulfhydryl. (R) represents a labeling reagent or one end of a crosslinker having the iodoacetyl or bromoacetyl reactive group; (P) Thiol-containing compounds, such as dithiothreitol represents a protein or other molecule that contains the target functional (DTT) and β-mercaptoethanol (BME (also known as group (i.e., sulfhydryl, –SH). 2-mercaptoethanol)), must be excluded from reaction Histidyl side chains and amino groups react in the buffers used with maleimides because they will compete unprotonated form with iodoacetyl groups above pH 5 for coupling sites. For example, if DTT were used to reduce and pH 7, respectively. To limit free iodine generation, disulfides in a protein to make sulfhydryl groups available for which has the potential to react with tyrosine, histidine, conjugation, the DTT would have to be thoroughly removed and tryptophan residues, perform iodoacetyl reactions using a desalting column before initiating the maleimide and preparations in the dark. Avoid exposure of iodoacetyl reaction. Interestingly, the disulfide-reducing agent TCEP compounds to reducing agents.

9 Pyridyl disulfides Carbonyls (aldehydes) as crosslinking targets

Pyridyl disulfides react with sulfhydryl Aldehydes (RCHO) and ketones (RCOR´) are reactive

S groups over a broad pH range to form varieties of the more general functional group called S N R disulfide bonds. As such, conjugates carbonyls, which have a carbon–oxygen double bond Pyridyldithiol prepared using these crosslinkers are (C=O). The polarity of this bond (especially in the context cleavable with typical disulfide-reducing of aldehydes) makes the carbon atom electrophilic and agents such as dithiothreitol (DTT). reactive to nucleophiles such as primary amines.

Pyridyl disulfide reaction chemistry Although aldehydes do not naturally occur in proteins Pyridyl disulfides react with sulfhydryl groups over a broad or other macromolecules of interest in typical biological pH range (the optimum is pH 4–5) to form disulfide bonds. samples, they can be created wherever oxidizable sugar During the reaction, a disulfide exchange occurs between groups (also called reducing sugars) exist. Such sugars are the molecule’s –SH group and the reagent’s 2-pyridyldithiol common monomer constituents of the polysaccharides or group. As a result, pyridine-2-thione is released and can carbohydrates in posttranslational glycosylation of many be measured spectrophotometrically (Amax = 343 nm) to proteins. In addition, the ribose of RNA is a reducing sugar. monitor the progress of the reaction. These reagents can be used as crosslinkers and to introduce sulfhydryl groups Periodic acid (HIO4) from dissolved sodium periodate into proteins. The disulfide exchange can be performed at (NaIO4) is a well-known mild agent for effectively oxidizing physiological pH, although the reaction rate is slower than vicinal diols in carbohydrate sugars to yield reactive in acidic conditions. aldehyde groups. The carbon–carbon bond is cleaved between adjacent hydroxyl groups. By altering the amount P of periodate used, aldehydes can be produced on a S SH S S N pH 6.5–7.5 S R + P R + S N smaller or larger selection of sugar types. For example, H Pyridyldithiol Sulfhydryl Cleavable conjugate Pyridine- treatment of glycoproteins with 1 mM periodate usually reagent on protein (disulfide bond) 2-thione affects only sialic acid residues, which frequently occur at the ends of polysaccharide chains. At concentrations Figure 9. Pyridyldithiol reaction scheme for cleavable (reversible) chemical conjugation to a sulfhydryl. (R) represents a labeling reagent of 6–10 mM periodate, other sugar groups in proteins will or one end of a crosslinker having the pyridyl disulfide reactive group; (P) be affected (Figure 10). represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH). Sodium meta-periodate Na+ O− O I O O O R´ O O O R´ Carbonyl-reactive chemical groups HO HO 10 mM R O * OH R O O OH O Carbonyl (–CHO) groups can be created in glycoproteins Internal mannose residue Aldehyde-activated by oxidizing the polysaccharide posttranslational O O modifications with sodium meta-periodate. Hydrazide OH OH NH NH OH and alkoxyamine reactive groups target aldehydes. These 1 mM HO OH HO + 2 CH O O ** O 2 O O aldehydes also react with primary amines to form Schiff O OH O O bases that can be further reduced to form a covalent bond R R Terminal sialic acid residue Aldehyde-activated (reductive amination). Figure 10. The reaction of sodium periodate with sugar residues Carbohydrate modification is particularly useful for creating yields aldehydes for conjugation reactions. R and R´ represent connecting sugar monomers of the polysaccharide. Red asterisks indicate target sites for conjugation on polyclonal antibodies sites of diol cleavage. Sialic acid is also called N-acetyl-D-neuraminic acid. because the polysaccharides are located in the Fc region. This results in labeling or crosslinking sites located away from antigen binding sites, ensuring that antibody function will not be adversely affected by the conjugation procedure.

10 Hydrazides Alkoxyamine reaction chemistry Alkoxyamine compounds conjugate to carbonyls to create Carbonyls (aldehydes and ketones) can be an oxime linkage (Figure 12). The reaction is similar to O produced in glycoproteins and other hydrazides, and can also use aniline as a catalyst. NH2 N R H polysaccharide-containing molecules by mild oxidation of certain sugar glycols using Hydrazide NH O N 2 pH 6.5–7.5 O R O + P R P sodium meta-periodate. Hydrazide-activated H H crosslinkers and labeling compounds will then conjugate Alkoxyamine Aldehyde Stable conjugate with these carbonyls at pH 5–7, resulting in formation of reagent (oxidized sugar) (oxime bond) hydrazone bonds. Figure 12. Alkoxyamine reaction scheme for chemical conjugation to an aldehyde. (R) represents a labeling reagent or one end of a crosslinker Hydrazide chemistry is useful for labeling, immobilizing, having the alkoxyamine reactive group; (P) represents a glycoprotein or or conjugating glycoproteins through glycosylation sites, other glycosylated molecule that contains the target functional group (i.e., an aldehyde formed by periodate oxidation of carbohydrate sugar groups, which are often (as with most polyclonal antibodies) such as sialic acid). located at domains away from the key binding sites whose function one wishes to preserve. Reductive amination

Hydrazide reaction chemistry In reductive amination, the electrophilic carbon atom of Aldehydes created by periodate oxidation of sugars in an aldehyde attacks the nucleophilic nitrogen of a primary biological samples react with hydrazides at pH 5–7 to amine to yield a weak bond called a Schiff base. Unlike form hydrazone bonds (Figure 11). Although this bond to a the bond formed with hydrazide or alkoxyamines, the hydrazide group is a type of Schiff base, it is considerably Schiff base formed with ordinary amines rapidly hydrolyzes more stable than a Schiff base formed with a simple (reverses) in aqueous solution and must be reduced to an amine. The hydrazone bond is sufficiently stable for most alkylamine (secondary amine) linkage to stabilize it. Sodium protein-labeling applications. If desired, however, the cyanoborohydride (NaCNBH3) is a mild reducing agent that double bond can be reduced to a more stable secondary performs this function effectively, without reducing other amine bond using sodium cyanoborohydride (see section chemical groups in biological samples (Figure 13). Like on reductive amination). carbodiimide crosslinking chemistry (carboxyl to amine), reductive amination (aldehyde to amine) is a zero-length O O crosslinking method. NH O N N 2 pH 6.5–7.5 N R H + P R H P H H Primary amine Sodium NH 2 cyanoborohydride Hydrazide Aldehyde Stable conjugate P NaCNBH3 reagent (oxidized sugar) (hydrazone bond) O O P P N O P P NH O P

Figure 11. Hydrazide reaction scheme for chemical conjugation to O O HO O HO O an aldehyde. (R) represents a labeling reagent or one end of a crosslinker R O OH R O OH R O OH having the hydrazide reactive group; (P) represents a glycoprotein or other Aldehyde groups Conjugate Stable conjugate glycosylated molecule that contains the target functional group (i.e., an (oxidized sugar groups) (Schi base bond) (secondary amine bond) aldehyde formed by periodate oxidation of carbohydrate sugar groups, such as sialic acid). Figure 13. Reductive amination, the conjugation of aldehydes and primary amines. The initial reaction results in a weak, reversible Schiff Alkoxyamines base linkage. Reduction with sodium cyanoborohydride creates a stable, irreversible secondary amine bond. Although not currently as popular or common

NH2 R O as hydrazide reagents, alkoxyamine compounds conjugate to carbonyls Alkoxyamine (aldehydes and ketones) in much the same manner as hydrazides.

11 Nonspecificly-reactive Aryl azide reaction chemistry chemical groups When an aryl azide is exposed to UV light (250–350 nm), it forms a nitrene group that can initiate addition reactions with double bonds, insertion into C–H and N–H sites, or Photoreactive crosslinkers are widely used for nonspecific subsequent ring expansion to react with a nucleophile (e.g., bioconjugation. While numerous options exist (Figure 14), primary amines). The latter reaction path dominates when the two most common photoreactive chemical groups primary amines are present in the sample (Figure 15). are diazirines and aryl azides. Photoreactive groups are activated by UV light and can be used in vitro and in vivo. Thiol-containing reducing agents (e.g., DTT or β-mercaptoethanol) must be avoided in the sample solution during all steps before and during photoactivation, because OH N HO N N+ N N+ they reduce the azide functional group to an amine, N− N+ N− N− preventing photoactivation. Reactions can be performed in a variety of amine-free buffer conditions. If working Phenyl azide Ortho-hydroxyphenyl azide Meta-hydroxyphenyl azide with heterobifunctional photoreactive crosslinkers, use

F − buffers compatible with both reactive chemistries involved. O O O F N N+ Experiments must be performed in subdued light and/ N+ N+ N N− N −O N+ N+ N− or with reaction vessels covered in foil until photoreaction F N− is intended. Typically, photoactivation is accomplished F Tetrafluorophenyl azide Ortho-nitrophenyl azide Meta-nitrophenyl azide with a handheld UV lamp positioned close to the reaction solution and shining directly on it (i.e., not through glass or polypropylene) for several minutes. OO N N+ NN N− O Three basic forms of aryl azides exist: simple phenyl O azides, hydroxyphenyl azides, and nitrophenyl O Diazirine Azido-methylcoumarin Psoralen azides. Generally, short-wavelength UV light (e.g., 254 nm; 265–275 nm) is needed to efficiently activate Figure 14. Common photoreactive chemical groups used for bioconjugation. simple phenyl azides, while long UV light (e.g., 365 nm; 300–460 nm) is sufficient for nitrophenyl azides. Because Aryl azides short-wavelength UV light can be damaging to other molecules, nitrophenyl azides are usually preferable for Photoreactive reagents are chemically crosslinking experiments. N N+ N– inert compounds that become reactive R UV Ring Nucleophile when exposed to ultraviolet or visible N N: + light expansion R–NH2 N − Phenyl azide light. Historically, aryl azides (also called N N N

phenyl azides) have been the most Phenyl azide Nitrene Dehydroazepine HN R reagent formed intermediate R–H popular photoreactive chemical group used in crosslinking R R–NH2 Reactive R–H hydrogen and labeling reagents. N H H N N N R R R R N H Photoreactive reagents are most often used as Addition Active hydrogen Active hydrogen heterobifunctional crosslinkers to capture binding partner reactions (C-H) insertion (N-H) insertion interactions. A purified bait protein is labeled with the Figure 15. Aryl azide reaction scheme for light-activated crosslinker using the amine- or sulfhydryl-reactive end. photochemical conjugation. Squiggle bonds represent a labeling Then this labeled protein is added to a lysate sample and reagent or one end of a crosslinker having the phenyl azide reactive group; allowed to bind its interactor. Finally, photoactivation with (R) represents a protein or other molecule that contains nucleophilic or active hydrogen groups. Bold arrows indicate the dominant pathway. UV light initiates conjugation via the phenyl azide group. Halogenated aryl azides react directly (without ring expansion) from the activated nitrene state.

12 Diazirines Chemoselectivity of azide–alkyne reactions Invitrogen™ Click-iT™ technology use azides and alkynes Diazirines are a newer class of photoactivatable as specific binding moieties in a two-step procedure that NN chemical groups that are being used in first labels and then subsequently detects the molecule of R crosslinking and labeling reagents. The diazirine interest. Azides and alkynes can be used interchangeably Diazirine (azipentanoate) moiety has better photostability in “click” reactions, so either one can serve as the tagged than phenyl azide groups, and it is more easily substrate or be labeled for the detection step. and efficiently activated with long-wave UV light (330–370 nm). Because azides and alkynes are extremely small and biocompatible moieties, these molecules can be Diazirine reaction chemistry incorporated into biological systems without disrupting Photoactivation of diazirine creates reactive carbene cellular function and incorporate normally into nucleic intermediates (Figure 16). Such intermediates can acids, complex polysaccharides, and proteins. form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at If an azide is incorporated into a cellular protein, it can be distances corresponding to the spacer arm lengths of labeled using an alkyne tagged with an Invitrogen™ Alexa the particular reagent. Diazirine analogs of amino acids Fluor™ dye. Conversely, if an alkyne is incorporated into a can be incorporated into protein structures by translation, cellular protein, it can be labeled using an azide tagged enabling specific recombinant proteins to be activated as with an Alexa Fluor dye. The mild reaction conditions of the the crosslinker. Click-iT Plus reagent help preserve the activity of fluorescent proteins, so click reactions can be multiplexed H Rʹ P Rʹ P NN UV light H with GFP or R-PE. : 350 nm R R R Chemoselectivity of azide–phosphine reactions Diazirine N2 Carbene Stable conjugate reagent formed (insertion bond) The Staudinger reaction occurs between a methyl ester phosphine (PH ) and an azide (N –) to produce an aza-ylide Figure 16. Diazirine reaction scheme for light-activated 3 3 photochemical conjugation. (R) represents a labeling reagent or one end intermediate that is trapped to form a stable covalent bond of a crosslinker having the diazirine reactive group; (P) represents a protein (Figure 17). or other molecule that contains nucleophilic or active hydrogen groups (Rʹ). The Staudinger ligation involves the reaction of azido Chemoselective ligation bioorthogonal probes with phosphine compounds. Similar to click chemistry, the ligation reaction is highly Chemoselective ligation specific and can be performed in aqueous environments Phosphine O refers to the use of at physiological pH. Staudinger ligations do not require H OCH3 N3 P N mutually specific pairs copper to be reactive. Although this increases the R P Azide Ph O Ph of conjugation reagents. biocompatibility of Staudinger ligations, these reactions Unlike typical crosslinking also tend to be slower than click reactions because of the methods used in biological research, this reaction absence of a catalyst. chemistry depends upon a pair of unique reactive O O N3 P P groups that are specific to one another and also foreign H OCH3 H N N Azide-labeled N H R P target molecule R P O Ph Ph to biological systems. Because these reactions O Ph O Ph Phosphine-activated Conjugate O (azide–alkyne or azide–phosphine) do not occur in compound (amide bond) H2O H OCH3 cells, these functional groups react only with each other N R P N P - Ph CH3O in biological samples, thus resulting in minimal background O Ph Aza-ylide intermediate and few artifacts, hence the term “chemoselective”. Figure 17. Staudinger ligation reaction scheme (azide–phosphine These specialized forms of crosslinking are most often conjugation). Phosphine-activated proteins or labeling reagents react with used for in vivo metabolic labeling. azide-labeled target molecules to form aza-ylide intermediates that quickly rearrange in aqueous conditions to form stable amide bonds between reactant molecules.

13 Molecular properties of bioconjugation reagents

Introduction Bioconjugation reagents are selected on the basis of their chemical reactivities and other chemical properties that affect their behavior in different applications. Key considerations include the chemical specificity of the reactive ends, reaction conditions, and if further modification to the protein or peptide of interest is required to enable bioconjugation. Other important factors that influence the functionality, specificity, and solubility of the bioconjuation reaction include the spacer arm length, cleavability, composition, and structure (Table 3).

14 Table 3. Molecular properties of bioconjugation reagents. O O O O N Chemical specificity The reactive target(s) of the crosslinker’s N O reactive ends. A general consideration O O O is whether the reagent has the same or different reactive groups at either DSS end (termed homobifunctional and Disuccinimidyl suberate heterobifunctional, respectively). MW 368.34 Spacer arm 11.4 Å General reaction The buffer system required to perform conditions bioconjugation. Variables include pH, buffer concentration, and protein Figure 18. Homobifunctional crosslinker example. DSS is a popular, concentration. simple crosslinker that has identical amine-reactive NHS ester groups at either end of a short spacer arm. The spacer arm length (11.4 Å) is the Modifications These specialized reagents add molecular final maximum molecular distance between conjugated molecules (i.e., mass, create a new functional group nitrogens of the target amines). that can be targeted in a subsequent reaction step, or increase the solubility of the molecule. Heterobifunctional crosslinkers possess different reactive Spacer arm length The molecular span of a crosslinker groups at either end (Figure 19). These reagents not only (i.e., the distance between conjugated allow single-step conjugation of molecules that have molecules). A related consideration is whether the linkage is cleavable or the respective target functional groups, but they also reversible. allow sequential (two-step) conjugations that minimize Spacer arm composition The chemical groups found within the undesirable polymerization or self-conjugation. In spacer arm. sequential procedures, heterobifunctional reagents are Spacer arm cleavability The availability of a cleavage site within the spacer arm between the chemical reactive reacted with one protein using the most labile group of groups. the crosslinker first. After removing excess unreacted Chain structure The presence of a straight or branched crosslinker, the modified first protein is added to a solution and solubility chain that can effect whether a crosslinker or modifier can permeate into cells and/ containing the second protein where reaction through or crosslink hydrophobic proteins within the second reactive group of the crosslinker occurs. The membranes. These properties are determined by the composition of the most widely used heterobifunctional crosslinkers are those spacer arm and/or reactive group. having an amine-reactive succinimidyl ester (NHS ester) at one end and a sulfhydryl-reactive group (e.g., maleimide) on the other end. Because the NHS ester group is less Homobifunctional and stable in aqueous solution, it is usually reacted to one heterobifunctional crosslinkers protein first. If the second protein does not have available native sulfhydryl groups, they can be added in a separate Crosslinkers can be classified as homobifunctional or prior step using sulfhydryl-addition reagents. heterobifunctional. Homobifunctional crosslinkers have O identical reactive groups at either end of a spacer arm O N + - (Figure 18). Generally, they must be used in one-step Na O O O S N O reaction procedures to randomly “fix” or polymerize O O molecules containing like functional groups. For example, O adding an amine-to-amine crosslinker to a cell lysate will Sulfo-SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate result in random conjugation of protein subunits, interacting MW 436.37 proteins, and any other polypeptides whose lysine side Spacer arm 8.3 Å chains happen to be near each other in the solution. This Figure 19. Heterobifunctional crosslinker example. Sulfo-SMCC is a is ideal for capturing a “snapshot” of all protein interactions popular crosslinker that has an amine-reactive sulfo-NHS ester group (left) but cannot provide the precision needed for other types at one end and a sulfhydryl reactive maleimide group (right) at the opposite end of a cyclohexane spacer arm. This allows sequential, two-step of crosslinking applications. For example, when preparing conjugation procedures. an antibody–enzyme conjugate, the goal is to link one to several enzyme molecules to each molecule of antibody without causing any antibody-to-antibody linkages to form. This is not possible with homobifunctional crosslinkers. Learn more at thermofisher.com/proteincrosslinking

15 Certain reagents have three termini, and are referred to as two components are difficult to analyze and provide less trifunctional crosslinkers or label transfer reagents. These information on spatial arrangements of protein subunits. compounds typically possess two chemically reactive functional groups and one label such as a biotin group. Table 4. Summary of characteristics of reactive groups. A commonly used example is sulfo-SBED (Figure 20). Optimal Reactive Target reaction group molecule Reaction buffer pH range - N Amine Aldehydes PBS (non-amine) p H 7.2 N+ O (oxidized N 21.2 Å NH carbohydrates) MES p H 4.5 –7.2 HN O Carboxylic acid H HN N (EDC-modified) S 14.3 Å O HN O Aryl azide Unsubstituted PBS (non-amine) aryl azides react primarily with S 24.7 Å S amines Carbodiimide Carboxylic acids, MES or PBS p H 4.5 –7.2 O O Sulfo-SBED hydroxyls MW 879.98 O N O Hydrazide Aldehydes, 0.1 M Na-acetate/ pH 5.5–7.5

O ketones phosphate S MES/non-amine p H 4.5 –7.2 + - O Na O Carboxylic acid buffer Up to pH 7.5 (EDC-modified) Figure 20. Trifunctional crosslinker example: sulfo-SBED. Imidoester Amines PBS, borate, pH 8–9 carbonate/ bicarbonate, HEPES General reaction conditions Iodoacetyl Sulfhydryls PBS, borate, pH 7.5–8.5 carbonate/ bicarbonate, HEPES In many applications, it is necessary to maintain the native Isocyanate Hydroxyls Nonaqueous structure of the protein complex, so crosslinking is most (PMPI) Amines often performed using near-physiological conditions. Maleimide Sulfhydryls Thiol-free pH 7 optimal Reaction buffers for commonly used reactive groups are pH 6.5–7.5 NHS ester Amines PBS, borate, pH 7.5 optimal summarized in Table 4. Optimal crosslinker-to-protein carbonate/ p H 7.2– 8.5 molar ratios for reactions must be determined empirically, bicarbonate, HEPES although product instructions for individual reagents Pyridyl Sulfhydryls Thiol-free pH 7–8 generally contain guidelines and recommendations for disulfide Vinyl sulfone Sulfhydryls Thiol-free pH 8 common applications. (HBVS)

Depending on the application, the degree of conjugation is an important factor. For example, when preparing Modifications immunogen conjugates, a high degree of conjugation is desired to increase the immunogenicity of the antigen. Protein analysis and detection techniques often require However, when conjugating to an antibody or an enzyme, more than direct conjugation with a bifunctional crosslinker a low to moderate degree of conjugation may be optimal or activated labeling reagent. For example, in many so that biological activity of the protein is retained. situations, specialized protein modifications are needed to add molecular mass, increase solubility for storage, or The number of functional groups on the protein’s surface create a new functional group that can be targeted in a is also important to consider. If there are numerous subsequent reaction step. Protein modification reagents target groups, a lower crosslinker-to-protein ratio can be are chemicals that block, add, change, or extend the used. For a limited number of potential targets, a higher molecular reach of functional groups. crosslinker-to-protein ratio may be required. Furthermore, the number of components should be kept low or to a Protein sulfhydryls (side chain of cysteine) are important minimum because conjugates consisting of more than regulators of protein structure and function. Reducing

16 agents are used to prevent intra- and intermolecular Thermo Scientific™ Pierce™ Citraconic Anhydride (Cat. disulfide bonds from forming between cysteine residues No. 20907) is used. Sulfo-NHS acetate is typically used of proteins in order to enable crosslinking or modification. to prevent polymerization when performing protein This reduction is sometimes carried out under denaturing crosslinking reactions and when conjugating peptides conditions to enhance reactivity of inaccessible to carrier proteins for immunogen production. Blocking disulfide bonds. Dithiothreitol (DTT), β-mercaptoethanol amines on the peptide allows directed conjugation of (BME), and Tris(2-carboxyethyl)phosphine hydrochloride carboxylic acids on the peptide to primary amines on the (TCEP·HCl) are frequently used to reduce the disulfide protein using Thermo Scientific™ EDC (Cat. No. 29980). bonds of proteins. TCEP·HCl is a potent, versatile, O + – Na O O odorless, thiol-free reducing agent with broad application S O N to protein and other research involving reduction of O O disulfide bonds (Figure 21). This unique compound is easily O soluble and very stable in many aqueous solutions. TCEP Sulfo-NHS acetate reduces disulfide bonds as effectively as dithiothreitol MW 259.17 (DTT), but unlike DTT and other thiol-containing reducing Figure 23. Sulfo-NHS acetate is a protein modification reagent for agents, TCEP does not have to be removed before certain blocking primary amines. sulfhydryl-reactive crosslinking reactions. SATA and related reagents contain an amine-reactive O OH O OH group and a protected sulfhydryl group. By reacting the SH RS O R + + O HO P OH R S HH HO P OH + compound to a purified protein, the side chain of lysine HS R O O O O residues can be modified to contain a sulfhydryl group TCEP for targeting with sulfhydryl-specific crosslinkers or Figure 21. TCEP reduces disulfide bonds within bioconjugation immobilization chemistries. The method does not actually reagents and proteins. convert the amine into a sulfhydryl; rather, it attaches a sulfhydryl-containing group to the primary amine. The Chaotropic and denaturing chemical agents, including urea effect is also to extend the length of the side chain by and guanidine hydrochloride, disrupt water interactions and several angstroms. promote hydrophobic protein and peptide solubilization, elution, refolding, and structural analysis. O O S + – NH2 Cl N S Certain reagents are capable of reacting permanently or O O O Traut’s Reagent reversibly with sulfhydryl groups (e.g., NEM or MMTS, MW 137.63 SATA Spacer arm 8.1 Å respectively). These reagents add a very small “cap” on the MW 231.23 native sulfhydryl, enabling the activity of certain enzymes to Spacer arm 2.8 Å be controlled for specific assay purposes (Figure 22). Figure 24. Sulfhydryls can be converted to amines using SATA or Traut’s Reagent. O

CH3 N OS Chemically attaching single- or branched-chain S CH3 H C O polyethylene glycol (PEG) groups to proteins is a form of O 3 NEM MMTS labeling or modification that is primarily used to confer N-Ethylmaleimide MW 126.20 MW 125.13 water solubility and/or inert molecular mass to proteins. Forms of PEG that have been synthesized to contain Figure 22. Sulfhydryls can be blocked using NEM and MMTS. reactive chemical groups comprise ready-to-use, activated reagents for PEGylation (Figure 25). Sulfo-NHS acetate is a protein modification reagent that reacts with primary amines at pH 7.0–9.0, allowing modifications to occur in many standard non–amine- containing buffers. Once reacted, the amine is irreversibly Learn more at capped with an acyl group. For reversible amine blocking, thermofisher.com/proteinmodification

17 O O crosslinking because they are hydrophobic and uncharged.

N CH3 O O For example, if a charged sulfonate group is added to the MS(PEG)n Methyl-PEG -NHS ester O n n termini of a hydrophobic crosslinker, a water-soluble analog Succinimidyl-((N-methyl)-#ethyleneglycol) ester n = 8 is formed. A good example of this is the comparison of MS(PEG)8 3 16.4 Å MW 509.4 DSS with BS . DSS is soluble in organic solvents whereas O Spacer arm 30.8 Å 3 O BS is soluble is aqueous buffers. BS(PEG)5 is also water- N O O n = 12 O O O CH 3 MS(PEG)12 soluble because of its PEG spacer (Figure 27). O MW 685.71 MS(PEG) Spacer arm 44.9 Å 4 O MW 333.33 n = 24 O O Spacer arm 16.4 Å O N MS(PEG)24 N O MW 1,214.39 O O Spacer arm 88.2 Å O DSS Figure 25. Examples of single-chain, amine-reactive Disuccinimidyl suberate PEGylation reagents. MW 368.34 Spacer arm 11.4 Å O Na+O– O O – + O S N O O Na Spacer arm length O O N S O O O O O BS3 The spacer arm is the chemical chain between two reactive Bis(sulfosuccinimidyl) suberate MW 572.43 groups or between a reactive group and a label. The length Spacer arm 11.4 Å of a spacer arm (measured in Å) determines how flexible O O O O N O O N a conjugate will be. Longer spacer arms have greater O O O O O flexibility and reduced steric hindrance. Longer spacer O O BS(PEG)5 arms have the caveat of possessing more sites for potential Bis(succinimidyl) penta(ethylene glycol) MW 532.50 nonspecific binding. Spacer arms can range from zero Spacer arm 21.7 Å length to >100 Å (Figure 26). Figure 27. Crosslinkers with various spacer arm compositions.

O FF O O O N –O O N O N+ N+ O O Spacer arm cleavability O O– O DFDNB DSS 1,5-difluoro-2,4-dinitrobenzene Disuccinimidyl suberate MW 204.09 MW 368.34 Crosslinkers and protein modification reagents form Spacer arm 3.0 Å Spacer arm 11.4 Å stable, covalent bonds with the proteins they react with.

O O O O In certain applications, it is desirable to have the ability to N O O O O N O O O O O O O break that bond and recover the individual components. O O Bioconjugation reagents are available with a cleavage BS(PEG)9 Bis(succinimidyl) nona(ethylene glycol) MW 708.71 site built into the spacer arm. The most commonly used Spacer arm 35.8 Å cleavage site is a disulfide bridge, which can be readily Figure 26. Different lengths of spacer arms. reduced with the introduction of a common reducing agent such as β-mercaptoethanol, dithiothreitol, or TCEP. An Spacer arm composition example of this is the crosslinker DTBP (Figure 28). + – NH2 Cl O S The molecular composition of a bioconjugation spacer arm S O NH +Cl– can affect solubility and nonspecific binding. Traditional 2 DTBP crosslinkers and labeling reagents have spacer arms that Dimethyl 3,3' dithiobispropionimidate·2HCl contain hydrocarbon chains or polyethylene glycol (PEG) MW 309.28 Spacer arm 11.9 Å chains. Hydrocarbon chains are not water-soluble and typically require an organic solvent such as DMSO or Figure 28. The disulfide bridge built into the spacer arm of DTBP allows easy cleavage of a protein conjugate using standard DMF for suspension. These reagents are better suited for reducing agents. penetrating the cell membrane and performing intercellular

18 Spacer arm structure and solubility

Crosslinkers typically possess a straight chain spacer arm, Some crosslinkers contain a spacer arm formed from PEG but protein modification reagents allow more options. A subunits, resulting in a polyethylene oxide (PEO) chain with good example is Thermo Scientific™ PEGylation reagents abundant oxygen atoms to provide water solubility. These that can be either straight or branched. For example, crosslinkers are designated by a (PEG)n in their name and CA(PEG) is a straight-chain PEGylation reagent and are both water-soluble and unable to penetrate biological

TMM(PEG)12 is a branched reagent (Figure 29). membranes. They provide the added benefit of transferring their hydrophilic spacer to the crosslinked complex, Many crosslinkers, by virtue of their hydrophobic spacer decreasing the potential for aggregation and precipitation arms, have limited solubility in aqueous solutions. These of the complex. crosslinkers are generally dissolved in DMF or DMSO, then added to the biological system or solution of biomolecules Other crosslinkers obtain their water solubility and their to be crosslinked. Hydrophobic crosslinkers are able ability to permeate the membrane by virtue of a charged to cross cellular and organellar membranes and affect reactive group at either end of the spacer. These charged crosslinking both at the outer surface of a membrane reactive groups, such as sulfo-NHS esters or imidoesters, and within the membrane-bound space. It is often impart water solubility to the crosslinking reagent, but not inconvenient or undesirable to introduce organic solvents to the crosslinked complex because the reactive group is into a crosslinking procedure for a biological system. It is not a part of the final complex. also desirable in many instances to affect crosslinking only on the outer surface of a cellular or organellar membrane without altering the interior of the cell or organelles. For such cases, several water-soluble, membrane-impermeant crosslinkers are available.

O

N

O O

HN

O TMM(PEG)12 (Methyl-PEG12)3-PEG4-maleimide MW 2,360.75 O 27.6 Å O O 52.0 Å NH2 HO O O CA(PEG)n n O O O O O O O CH3 Carboxy-PEGn-amine N O O O O O O H Carboxyl-(ethyleneglycol)n ethylamine n = 8 O O CA(PEG)8 HN H MW 441.51 O N O O O O O O O O O O O O CH 18.1Å Spacer Arm 33.6Å 3 O O O n = 12 O O CA(PEG)12 O O O O O O CH3 HO O O NH2 O N O O O O O O MW 617.72 H Spacer Arm 46.8Å CA(PEG)4 MW 265.30 n = 24 Spacer Arm 18.1Å CA(PEG) 24 46.2 Å MW 1146.35 Spacer Arm 89.8Å Figure 29. Straight and branched protein modification reagents.

19 Applications using bioconjugation reagents

Introduction Bioconjugation reagents have a variety of applications in life science research and assay development. These include protein and peptide biotinylation, fluorescent labeling of proteins and peptides, protein immobilization onto solid supports, hapten–carrier conjugation, protein– protein conjugation, creation of immunotoxins, label transfer, subunit crosslinking and protein structural studies, protein interaction and crosslinking using mass spectrometry, in vivo crosslinking, metabolic labeling, cell surface crosslinking, and cell membrane structural studies. Protein conjugates are often designed to enable purification and detection in a complex biological sample. Commonly, antibodies are the target of bioconjugation with common labels such as biotin or chemically reactive fluorescent dyes. In this process, NHS ester chemistry is the most widely used method for labeling available lysine residues.

20 Protein and peptide biotinylation these reactive groups, biotin can be easily attached to most proteins and peptides. The highly specific interaction of avidin with biotin (vitamin H) can be a useful tool in designing nonradioactive Biotinylation reagents are available for targeting a purification and detection systems. The extraordinary variety of functional groups, including primary amines, 15 –1 affinity of avidin for biotin (Ka = 10 M ) is the strongest sulfhydryls, carbohydrates, and carboxyls. Photoreactive known noncovalent interaction of a protein and ligand, and biotin compounds that react nonspecifically upon allows biotin-containing molecules in a complex mixture to photoactivation are also available. This variety of functional be discretely bound with avidin conjugates. Desthiobiotin group specificities is extremely useful, allowing the choice is a modified form of biotin that binds less tightly to of a biotinylation reagent that does not inactivate the avidin and streptavidin than biotin while still providing target macromolecule. excellent specificity in affinity purification methods. Unlike biomolecules that are labeled with biotin, proteins and Several cleavable or reversible biotinylation reagents other targets that are labeled with desthiobiotin can be are also available and allow specific elution of the eluted without harsh denaturing conditions (Figure 30). biotinylated molecule from biotin-binding proteins and peptides. The most frequently used biotinylation O O reagents, N-hydroxysuccinimide (NHS) esters and N-hydroxysulfosuccinimide (sulfo-NHS) esters, react with HN NH NH HN primary amines (Figure 31). While NHS esters of biotin are the most frequently used biotinylation reagents, they are HO HO S Remove not necessarily the best for a particular application. If only O sulfur O a portion of the primary amines on a protein are reacted, Biotin MW 244.31 reaction with NHS esters of biotin will result in a random Desthiobiotin distribution of biotin on the surface of the protein. If a MW 214.26 particular primary amine is critical to the biological activity Figure 30. Comparison of the chemical structures of biotin of the protein, modification of this critical amine may result and desthiobiotin. in the loss of its biological activity. Depending on the extent The extensive line of Thermo Scientific™ biotinylation of biotinylation, complete loss of activity may occur. labeling reagents exploits this unique interaction. Biotin, a 244 dalton vitamin found in tiny amounts in all living cells, Antibodies are biotinylated more often than any other binds with high affinity to avidin-based molecules. Since class of proteins, and it is advantageous to biotinylate biotin is a relatively small molecule, it can be conjugated to in a manner that will maintain immunological reactivity. many proteins without significantly altering their biological Thermo Scientific™ Sulfo-NHS-LC-Biotin is an excellent activity. The valeric acid side chain of the biotin molecule choice for labeling both monoclonal and polyclonal can be derivatized to incorporate various reactive groups antibodies because it is the simplest and often the most that are used to attach biotin to other molecules. Using effective method.

O O– – O Sulfo-NHS-LC-biotin O O S NH S Sulfo-NHS leaving group O O HN O O O (remove by desalting) H N N N O S OH O O O O Biotinylated molecule NH HN NH Protein with 2 O P P H primary amines N N S H O Figure 31. Biotinylation using NHS ester chemistry.

21 Understanding the functional groups available on Fluorescent labeling of proteins an antibody is the key to determining a strategy for and peptides modification.

• Primary amine groups (–NH ) are found on lysine 2 Fluorescent molecules, also called fluorophores, respond side chains and at the amino terminus of each directly and distinctly to light and produce a detectable polypeptide chain. signal. Unlike enzymes or biotin, fluorescent labels do • Sulfhydryl groups (–SH) can be generated by reducing not require additional reagents for detection. This feature disulfide bonds in the hinge region. makes fluorophores extremely versatile and the new standard in detecting protein location and activation, • Carbohydrate residues containing cis-diols can be identifying protein complex formation and conformational oxidized to create active aldehydes (–CHO). changes, and monitoring biological processes in vivo. The vast selection of fluorophores today provides The fast and reliable procedure has been optimized for greater flexibility, variation, and fluorophore performance antibody modification. If the antibody contains a lysine-rich for research applications than ever before. Reactive antigen-binding site, amine-reactive reagents may inhibit fluorescent dyes are used to label biomolecules for antigen binding. Traditional strategies were developed detection by fluorescence microscopy, flow cytometry, using biotin derivatives that react with sulfhydryl or high-content analysis, high-density arrays, in vivo imaging, aldehyde groups. immunoblotting, and immunostaining. Fluorescent dyes are available activated with NHS ester chemistry or A simple alternative to these traditional methods is the maleimide chemistry for labeling at primary amines and SiteClick™ antibody labeling system (Figure 32), which reduced sulfhydryls, respectively. Additionally, a wide range allows simple and gentle site-selective attachment of of fluorescent derivatives are available for biorthogonal detection molecules to heavy-chain N-linked glycans— labeling and click chemistry, and for modifying alcohols, far from the antigen-binding domain. This method also aldehydes, and ketones. Key considerations in selecting provides excellent reproducibility from labeling to labeling reactive dyes are brightness, photostability, instrument and from antibody to antibody because the N-linked compatibility, color selection, pH insensitivity, and glycans are highly conserved. The simple methodology is water solubility. based on the enzyme- and copper-free click chemistry. In addition to biotin, a number of different detection molecules can be site-selectively attached to the heavy- chain glycans—including phycobiliproteins (e.g., R-PE), Invitrogen™ Qdot™ probes, Alexa Fluor dyes, and metal- chelating compounds.

Antibody β-Galactosidase • Polyclonal GalT(Y289L) DIBO alkyne label • Monoclonal UDP-GalNAz • Recombinant

N2 N2 N2 N2

Azide-activated antibody Site-selectively labeled antibody Figure 32. The SiteClick antibody labeling system. The first step in the SiteClick antibody labeling process involves removal of terminal galactose residues from the heavy-chain N-linked glycans using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor 488 DIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody.

Learn more at thermofisher.com/siteclick

22 Table 5. Overview of reactive fluorescent dyes.

Emission Amine- Thiol- Chemoselective –COOH or Ex/Em (nm) Fluorophore color reactive reactive (Click-iT) –NH2 terminated Blue 346/442 Alexa Fluor 350 • • •

Blue 402/421 Alexa Fluor 405 •

Blue 410/455 Pacific Blue • •

Blue 375/470 7-Aminocoumarin • •

Green 500/506 BODIPY 493/503 •

Green 505/513 BODIPY FL • • •

Green 494/518 Fluorescein (FITC) • • •

Green 495/519 Alexa Fluor 488 • • •

Green 505/520 iFL pHrodo Green • •

Green 495/524 Oregon Green 488 • • • •

Green 405/525 Qdot 525 • •

Yellow 511/530 Oregon Green 514 • •

Yellow 517/542 Alexa Fluor 514 •

Yellow 434/539 Alexa Fluor 430 •

Yellow 405/545 Qdot 545 •

Orange 528/550 BODIPY R6G •

Orange 400/551 Pacific Orange •

Orange 531/554 Alexa Fluor 532 • •

Orange 534/554 BODIPY 530/550 •

Orange 405/565 Qdot 565 • • •

Orange 555/565 Alexa Fluor 555 • • • •

Orange 558/569 BODIPY 558/568 •

23 Table 5. Overview of reactive fluorescent dyes. (continued)

Emission Amine- Thiol- Chemoselective –COOH or Ex/Em (nm) Fluorophore color reactive reactive (Click-iT) –NH2 terminated

Orange/red 542/574 BODIPY TMR • •

Orange/red 556/575 Alexa Fluor 546 • •

Tetramethylrhodamine Orange/red 555/580 • • • • (TMR; TRITC)

Orange/red 405/585 Qdot 585 • •

Red 570/590 Rhodamine Red (RITC) • •

Red 576/590 BODIPY 576/589 •

Red 566/590 iFL pHrodo Red • •

Red 578/603 Alexa Fluor 568 • •

Red 405/605 Qdot 605 • •

Red 580/605 X-Rhodamine (XRITC) • • •

Red 595/615 Texas Red • • •

Red 589/617 BODIPY TR •

Red 590/617 Alexa Fluor 594 • • •

Red 405/625 Qdot 625 • •

Far red 625/640 BODIPY 630/650 •

Far red 632/647 Alexa Fluor 633 • •

Far red 405/655 Qdot 655 • •

Far red 646/660 BODIPY 650/665 •

Far red 650/668 Alexa Fluor 647 • • •

Far red 679/702 Alexa Fluor 680 • •

NIR 405/705 Qdot 705 • •

NIR 702/723 Alexa Fluor 700 •

NIR 749/775 Alexa Fluor 750 • •

NIR 405/800 Qdot 800 • •

NIR 782/805 Alexa Fluor 790 •

Learn more at thermofisher.com/fluorophores

24 Protein immobilization onto too close to the matrix to allow access by the receptor. solid supports Steric effects are usually most pronounced when the ligand is a small molecule. The aldehyde-activated Thermo Scientific™ AminoLink™ Plus agarose resin Proteins, peptides, and other molecules can be (Cat. No. 20501) uses reductive amination, Thermo immobilized onto solid supports for affinity purification Scientific™ Pierce™ NHS-activated agarose (Cat. No. of proteins or for sample analysis. The supports may be 26200) uses NHS ester chemistry, and Thermo Scientific™ nitrocellulose or other membrane materials, polystyrene SulfoLink™ resin (Cat. No. 20401) uses haloacetyl chemistry plates or beads, agarose, beaded polymers, or glass to immobilize molecules, whereas Thermo Scientific™ slides. Some supports can be activated for direct coupling CarboxyLink™ resin (Cat. No. 20266) uses carbodiimide to a ligand. Other supports are made with nucleophiles or chemistry (Figure 33). other functional groups that can be linked to proteins using crosslinkers. Carbodiimides such as Thermo Scientific™ Thermo Scientific™ crosslinkers DMP (Cat. No. 21666) and EDC (Cat. No. 22980, 22981) are very useful for coupling DSS (Cat. No. 21555) are used to immobilize antibodies proteins to carboxy- and amine-activated glass, plastic, on Protein A or Protein G supports for antigen purification. and agarose supports. Carbodiimide procedures are After the antibody binds to the Fc-binding proteins, the usually one-step methods; however, two-step methods are antibody is oriented so that the Fab region is available possible if reactions are performed in organic solvents, or for antigen binding. DSS or DMP is applied to the bound if Thermo Scientific™ NHS (Cat. No. 24500) or Sulfo-NHS antibody column to link the two proteins through primary (Cat. No. 24510) is used to enhance the reaction. EDC is amines. Thermo Scientific™ Pierce™ Crosslink IP Kit useful for coupling ligands to solid supports and to attach (Cat. No. 26147) is based on this chemistry and utilizes leashes onto affinity supports for subsequent coupling of DSS to covalently immobilize the captured antibody to ligands. Useful spacers are diaminodipropylamine (DADPA), Protein A/G Agarose resin. The antibody resin is then ethylenediamine, hexanediamine, 6-aminocaproic acid, and incubated with the sample that contains the protein antigen any of several amino acids or peptides. Spacer arms help of interest, allowing the antibody–antigen complex to form. to overcome steric effects when the ligand is immobilized After washing, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit.

O H N Y Y ose 2 Y ar C + The entire procedure is performed in a microcentrifuge bead Y

Bead H H2N Bead Ag Agarose NH Y 2 Y NH2 Y spin cup, allowing solutions to be fully separated from the Aldehyde-activated Amine ligand Covalently agarose resin (antibody) immobilized antibody agarose resin upon brief centrifugation. Only the antigen

O is eluted by the procedure, enabling it to be identified O Y Y H2N ose N + Y ar Y

bead O Bead and further analyzed without interference from antibody

Bead H2N Ag Agarose NH Y O 2 Y NH2 Y fragments. Furthermore, the antibody resin often can be NHS-activated Amineligand Covalently agarose resin (antibody) immobilized antibody reused for additional rounds of immunoprecipitation.

H H

ose N ose N + HS ar I Peptide ar S bead bead Peptide Bead Bead DSS crosslinker Agarose Ag Ag Agarose O O ose Protein ose Protein ar ar bead bead 12-atom Iodoacetyl Thioether + HI Bead A/G Bead A/G Ag Ag spacer group bond Agarose Agarose Reduced sulfhydryl SulfoLink coupling resin molecule Immobilized peptide

HO Peptide H H Figure 34. Covalent attachment of captured antibody to Protein A/G N N NH 2 + bead

Bead Agarose resin using DSS crosslinker.

Agarose Agarose O Agarose

Immobilized DADPA Carboxyl ligand (CarboxyLink Resin) (e.g., peptide C-terminus)

EDC crosslinker

H H H N N N Peptide bead Bead Agarose Agarose Agarose O

Covalently immobilized ligand (attached by amide bond and long spacer arm)

Figure 33. Immobilization of biomolecules onto solid supports using different bioconjugation chemistries.

25 Surface modification using Thiols readily bind to gold surfaces, forming dative bonds, PEG-based reagents and have been used extensively for the modification of various surfaces such as quantum dots, self-assembled monolayers, and magnetic particles. Monodentate thiols, Polyethylene glycol (PEG) compounds of discrete chain however, can be easily removed by compounds such length can provide linkers of known molecular dimension as DTT. Bidentate thiols, such as lipoamides, provide an for creating biocompatible planar surfaces or particles. In added level of stability to the nanostructure and are much particular, PEG reagents containing a carboxylate group more resistant to removal from the metal surface by DTT on one end and a thiol or lipoamide group on the other or similar reagents. In addition to their increased stability, are effective as hydrophilic bridges between an adsorptive bidentate thiols provide added flexibility owing to the fact surface and an affinity ligand. that they can be used with a variety of metal surfaces (e.g., gold or silver nanoparticles).

Combining CA(PEG)12 molecules with MA(PEG)8 derivatives

(or CT(PEG)12 with MT(PEG)8 as thiol reagents) in surface modification can form a hydrophilic lawn of methyl ether– terminated PEGs with periodic exposed carboxylic acid– containing PEGs (Figure 35). The exposed carboxylic acid groups can be coupled to affinity ligands using the carbodiimide coupling reaction with EDC and sulfo- NHS. In addition, functionalization of solid surfaces with polyethylene glycol spacers significantly reduces nonspecific protein binding.

HO HO O O

O O

O O

O O

O O

H3C H3C H3C H3C O O O O O O

O O O O O O

O O O O O O O O S O- O O O O O O O N O EDC/sulfo-NHS Methyl-PEG8-amine O OH O O O O O O O O Amino-PEG12-carboxylate

Surface or particle Amine-reactive O O O O O O containing carboxylates sulfo-NHS esters O O O O O O

O O O O O O

NH HN NH NH HN NH O O O O O O

PEGylated surface containing a “lawn” of mPEG molecules with longer carboxy-PEG chains sticking o for subsequent conjugation with amine-containing ligands

Figure 35. Shorter methyl-PEGn-amine reagents can be combined with longer PEG compounds containing terminal carboxylate groups to create the classic “flowers in the grass” surface modification. The carboxylate groups then can be used to immobilize affinity groups using a carbodiimide coupling procedure with EDC and sulfo-NHS.

Learn more at thermofisher.com/pegylation

26 Hapten–carrier conjugation for This efficient reaction produces a conjugated immunogen antibody production in less than 2 hours. Often peptides are synthesized with terminal cysteines to enable attachment to supports or to carrier proteins using sulfhydryl- and amine-reactive, Several approaches are available for conjugating haptens heterobifunctional crosslinkers. By reacting the reagent to carrier proteins. The choice of which conjugation first to the carrier protein (with its numerous amines) and chemistry to use depends on the functional groups then to a peptide containing a reduced terminal cysteine, available on the hapten, the required hapten orientation all peptide molecules can be conjugated with the same and distance from the carrier, and the possible effect of predictable orientation (Figure 37). This method can be very conjugation on biological and antigenic properties. For efficient and yield an immunogen that is capable of eliciting example, proteins and peptides have primary amines a good response upon injection. (the N-terminus and the side chain of lysine residues), carboxylic groups (C-terminus or the side chain of aspartic acid and glutamic acid), and sulfhydryls (side chain of Carrier NH2 + protein Na cysteine residues) that can be targeted for conjugation. – O O O Generally, it is the many primary amines in a carrier protein S O O N O N O that are used to couple haptens via a crosslinking reagent. O O Sulfo-SMCC crosslinker Many crosslinkers are used for making conjugates for use O Carrier NH N as immunogens. The best crosslinker to use depends on protein O the functional groups present on the hapten and the ability O Maleimide-activated carrier protein of the hapten–carrier conjugate to function successfully as HS Peptide an immunogen after its injection. Carbodiimides are good choices for producing peptide–carrier protein conjugates S O Peptide Carrier because both proteins and peptides usually contain several NH N protein O carboxyls and primary amines. Carbodiimides such as EDC O react with carboxyls first to yield highly reactive unstable Carrier-peptide conjugate intermediates that can then couple to primary amines Figure 37. Peptide conjugation to carrier proteins for (Figure 36). antibody production.

Primary amine – H H Cl N+ N+ H N+ H2N P O O P OH O N NH + N + C N C H O NH O NH C C N Carboxylic O-acylisourea Peptide-carrier Isourea acid EDC active ester conjugate by-product

Figure 36. EDC-mediated conjugation of peptides and carrier proteins. Carrier proteins (C) and peptides (P) have both carboxyls and amines, so conjugation occurs in both orientations. Carrier proteins are very large in comparison to typical peptide haptens; therefore, numerous conjugation sites exist on each carrier protein molecule.

Learn more at thermofisher.com/carrierproteins

27 Protein–protein conjugation Homobifunctional NHS ester or imidoester crosslinkers may be used in a one-step protocol, but polymerization and One of the most common applications for crosslinkers self-conjugation are also likely. Homobifunctional sulfhydryl- is the production of protein–protein conjugates. reactive crosslinkers such as Thermo Scientific™ Pierce™ Conjugates are often prepared by attachment of an BMH (Cat. No. 22330) may be useful if both proteins to enzyme, fluorophore, or other molecule to a protein that be conjugated contain sulfhydryls. Heterobifunctional has affinity for one of the components in the biological crosslinkers are perhaps the best choices for antibody– system being studied. Antibody–enzyme conjugates enzyme or other protein–protein crosslinking. Unwanted (primary or secondary antibodies) are among the most self-conjugation inherent when using homobifunctional common protein–protein conjugates used. Although NHS ester reagents or glutaraldehyde can be avoided secondary antibody conjugates are available and relatively by using a Thermo Scientific™ Pierce™ reagent such as inexpensive, enzyme-labeled primary antibodies are usually SMCC (Cat. No. 22360) or Sulfo-SMCC (Cat. No. 22322). expensive and can be difficult to obtain. Many reagents are Sulfo-SMCC is first conjugated to one protein, and the used for the production of antibody–enzyme conjugates. second is thiolated with SATA (Cat. No. 26102) or Traut’s Glutaraldehyde conjugates are easy to make, but they Reagent (Cat. No. 26101), followed by conjugation often yield conjugates that produce high background in (Figure 39). Alternatively, disulfides in the protein may be immunoassays. Carbohydrate moieties can be oxidized reduced, and the two activated proteins are incubated and then coupled to primary amines on enzymes in together to form conjugates free of dimers of either a procedure called reductive alkylation or amination. protein. Any of the other NHS ester, maleimide, or These conjugates often result in less background in pyridyl disulfide crosslinkers can be substituted for enzyme immunoassays and are relatively easy to prepare; sulfo-SMCC in this reaction scheme. Heterobifunctional however, some self-conjugation of the antibody may occur photoactivatable phenyl azide crosslinkers are seldom (Figure 38). used for making protein–protein conjugates because of low conjugation efficiencies.

Sodium meta-periodate Protein primary amines Sodium cyanoborohydride oxidizes glycoprotein sugars react with aldehydes reduces Schi bases to to reactive aldehyde groups to form Schi bases yield stable amine bonds

NH2 Ab NaIO4 NaCNBH3

HO O HRP O O HRP Ab N O HRP Ab NH O HRP

HO O O O HO O HO O

R O OH R O OH R O OH R O OH Glycoprotein Aldehyde Conjugate Stable conjugate sugars groups (Schi base bond) (amide bond)

Figure 38. Reaction scheme for labeling antibodies with enzymes such as HRP using reductive amination.

O

Enz NH2 Enz NH N HS Ab O Enzyme O Sulfhydryl- Maleimide-activated activated enzyme antibody

Na+ O– O O S S O O Ab O N O N Enz NH N O O O O O

Sulfo-SMCC crosslinker Enzyme–antibody conjugate

Figure 39. Reaction scheme for labeling reduced antibody fragments with maleimide-activated enzymes.

28 Creation of immunotoxins then be used to purify and/or detect the interacting protein. Label transfer is particularly valuable because of its ability Specific antibodies can be covalently linked to toxic to identify proteins that interact weakly or transiently with molecules and then used to target antigens on cells. the protein of interest. New non-isotopic reagents and Often these antibodies are specific for tumor-associated methods continue to make this technique more accessible antigens. Immunotoxins are brought into the cell by and simple to perform by any researcher. surface antigens and, once internalized, they proceed to kill the cell by ribosome inactivation or other means. The Label transfer reagents can also have biotin built into their type of crosslinker used to make an immunotoxin can structure. This type of design allows the transfer of a biotin affect its ability to locate and kill the appropriate cells. tag to an interacting protein after cleavage of a cross- For immunotoxins to be effective, the conjugate must be bridge. Thermo Scientific™ Pierce™ Sulfo-SBED reagent stable in vivo. In addition, once the immunotoxin reaches (Cat. No. 33033) is an example of such a trifunctional its target, the antibody must be separable from the toxin reagent (see Figure 20). It contains an amine-reactive to allow the toxin to kill the cell. Thiol-cleavable, disulfide- sulfo-NHS ester on one arm (built off the α-carboxylate containing conjugates have been shown to be more of the lysine core), a photoreactive phenyl azide group on cytotoxic to tumor cells than noncleavable conjugates of the other side (synthesized from the α-amine) and a biotin ricin A immunotoxins. Cells are able to break the disulfide handle (connected to the ε-amino group of lysine). The arm bond in the crosslinker, releasing the toxin within the containing the sulfo-NHS ester has a cleavable disulfide targeted cell. bond, which permits transfer of the biotin component to any captured proteins. Thermo Scientific™ Pierce™ SPDP (Cat. No. 21857) is a reversible NHS ester, pyridyl disulfide crosslinker used to In use, a bait protein first is derivatized with sulfo-SBED conjugate amine-containing molecules to sulfhydryls. For through its amine groups, and the modified protein is several years, this has been the “workhorse” crosslinker for allowed to interact with a sample. Exposure to UV light production of immunotoxins. The amine-reactive NHS ester (300–366 nm) couples the photoreactive end to the is usually reacted with the antibody first. In general, toxins nearest available C–H or N–H bond in the bait–prey do not contain surface sulfhydryls; therefore, sulfhydryls complex, resulting in covalent crosslinks between bait must be introduced into them by reduction of disulfides, and prey. Upon reduction and cleavage of the disulfide which is common for procedures involving ricin A chain spacer arm, the biotin handle remains attached to the and abrin A chain, or through chemical modification protein(s) that interacted with the bait protein, facilitating reagents. A second SPDP molecule can be used for this isolation or identification of the unknown species using purpose and is reacted with amines on the immunotoxin, streptavidin, Thermo Scientific™ NeutrAvidin™ Protein, or then reduced to yield sulfhydryls. monomeric avidin.

Another chemical modification reagent that is commonly The architecture of this trifunctional label transfer reagent used for production of immunotoxins is Thermo differs substantially from the bifunctional counterparts Scientific™ Pierce™ 2-Iminothiolane, also known as discussed above. The advantages become almost Traut’s Reagent (Cat. No. 26101). Traut’s Reagent immediately apparent just by examining the structure. reacts with amines and yields a sulfhydryl when its ring structure opens during the reaction. The reactive moieties are well-segregated within sulfo- SBED. Most importantly, with a biotin label designed Label transfer into sulfo-SBED, radiolabeling with 125I is no longer necessary. The biotin label can be used to significant Label transfer involves crosslinking interacting molecules advantage in a label transfer application. For example, (i.e., bait and prey proteins) with a labeled crosslinking biotin can operate as a handle for purification of the agent and then cleaving the linkage between bait and prey protein or prey protein fragments or as a detection prey such that the label remains attached to the prey. This target using streptavidin-HRP and colorimetric or method allows a label to be transferred from a known chemiluminescent substrates. protein to an unknown interacting protein. The label can

29 Applications of Sulfo-SBED in Protein Interaction Studies

Applications for sulfo-SBED B Since the first availability of this reagent in 1994, the number of literature references for use of sulfo-SBED in Protein 1 + sNHS – S-S ––––––– N 3 protein interaction–related applications has grown rapidly. Dark reaction Published applications show how sulfo-SBED can used to: pH 7.2

B • Define interactions of complexes with activator domains1 Protein 1 — S-S ––––––– N3 + Protein 2 • Clarify the mechanism of protein complex assembly2

UV 300–366 nm • Convert to a sulfhydryl-reactive trifunctional reagent to 5–15 minutes map interactions3

B • Study docking site and factor requirements for binding4 Protein 1 —– S-S ———— Protein 2 • Describe binding contacts of interactors5

6 • Confirm recognition of a specific phosphoepitope Reduce Digest with trypsin

• Search for putative binding partners7 B B • Gain insight into chaperone-mediated Protein 1 — SH + HS ——— Protein 2 —– S-S ———— refolding interactions8 SDS-PAGE Reduce • Investigate mechanism of protein interaction9

B • Facilitate receptor activity-directed affinity tagging (re-tagging)10 Protein 1 — SH + HS ——– Protein 2

• Detect low-abundance protein receptors Isolate biotin-containing sA fragment over immobilized • Find protein–carbohydrate interactions Western streptavidin or monomeric transfer avidin SH • Understand drug–receptor interactions11

sA B • Quantitate triple helix–forming oligonucleotides12

Elute and separate Routes to determining the prey protein identification using via reversed-phase HPLC sulfo-SBED are outlined schematically in Figure 40. Note Detect with streptavidin-HRP SH or anti-biotin Ab or Ab against that the biotin label is a purification handle for captured Protein 2 with HRP-labeled B prey protein. In the trypsin digestion strategy, the peptide(s) secondary Ab. trapped can offer information relating to the binding interaction interface. The biotin-labeled prey protein or prey protein peptides recovered as result of the strategies outlined below can be subjected to several detection and Western blot Sequence Mass spec identification options designed to discover the identity of the detection analysis identification prey protein.

Legend: sNHS S-S N3 B Sulfo N-hydroxy Disulfide bond Phenyl azide Biotin succinmide ester

Figure 40. Applications of sulfo-SBED in protein interaction studies.

30 Subunit crosslinking and protein For determination or confirmation of the three-dimensional structural studies structure, cleavable crosslinkers with increasing spacer arm lengths may be used to determine the distance between subunits. Experiments using crosslinkers with different Crosslinkers can be used to study the structure and reactive groups may indicate the locations of specific composition of proteins in samples. Some proteins amino acids. Once conjugated, the proteins are subjected are difficult to study because they exist in different to two-dimensional electrophoresis. In the first dimension, conformations with varying pH or salt conditions. One way the proteins are separated using nonreducing conditions to avoid conformational changes is to crosslink subunits. and the molecular weights are recorded. Some subunits Amine-, carboxyl-, or sulfhydryl-reactive reagents are may not be crosslinked and will separate according to their used for identification of particular amino acids or for individual molecular weights, while conjugated subunits determination of the number, location, and size of subunits. will separate according to the combined size. The second Short to medium spacer arm crosslinkers are selected dimension of the gel is then performed using conditions to when intramolecular crosslinking is desired. If the spacer cleave the crosslinked subunits. The individual molecular arm is too long, intermolecular crosslinking can occur. weights of the crosslinked subunits can be determined. Carbodiimides that result in no spacer arm, along with Crosslinked subunits that were not reduced will produce short-length conjugating reagents, such as amine-reactive a diagonal pattern, but the cleaved subunits will be off the Thermo Scientific™ Pierce™ DFDNB (Cat. No. 21525), can diagonal. The molecular weights of the individual subunits crosslink subunits without crosslinking to extraneous should be compared with predetermined molecular weights molecules if used in optimal concentrations and conditions of the protein subunits using reducing SDS-polyacrylamide (Figure 41). Slightly longer crosslinkers, such as Thermo gel electrophoresis. Scientific™ DMP (Cat. No. 21666, 21667), can also crosslink subunits, but they may result in intermolecular coupling. Adjusting the reagent amount and protein concentration can control intermolecular crosslinking. Dilute protein solutions and high concentrations of crosslinker favor intramolecular crosslinking when homobifunctional crosslinkers are used.

FF

O NH +Cl– NH +Cl– –O 2 2 N+ N+ O O O O– DFDNB DMP 1,5-difluoro-2,4-dinitrobenzene Dimethyl pimelimidate·2HCl MW 204.09 MW 259.17 Spacer arm 3.0 Å Spacer arm 9.2 Å

Figure 41. DFDNB and DMP are used for crosslinking between protein subunits.

31 Protein interaction and crosslinking Our MS-grade crosslinkers are high-quality reagents that using mass spectrometry are available in multiple packaging options and sizes. We offer extensive technical expertise and support for various applications, as well as validation of these products in Chemical crosslinking in combination with mass workflows using Thermo Scientific™ mass spectrometers. spectrometry is a powerful method to determine protein– protein interactions. This method has been applied to Highlights recombinant and native protein complexes, and more • High quality—products manufactured in ISO 9001– recently, to whole cell lysates or intact unicellular organisms certified facilities in efforts to identify protein–protein interactions on a ™ global scale. • Convenience—products available in No-Weigh packaging or in multiple pack sizes

™ Thermo Scientific MS-grade crosslinkers are available with • More choices—available with different linker lengths, different linker lengths and as isotopically labeled sets to MS cleavability, and deuterium isotope labels help elucidate protein–protein interactions (Table 6). Both traditional noncleavable and MS-cleavable crosslinkers • Technical support—extensive web resources and can provide insight into the identification of protein– support to help ensure successful results protein interaction sites. These high-quality reagents have been validated in protein–protein interaction studies using Thermo Scientific™ mass spectrometers utilizing different types of fragmentation (CID, HCD, ETD, and EtHCD) and levels of tandem mass spectrometry (MS2 and MS3), in order to improve identification of protein–protein interaction sites.

Table 6. Overview of Thermo Scientific crosslinkers used for studying protein–protein interactions.

3 3 Crosslinker DSS BS BS -d4 DSG

O + – O O O O O O Na O Na+O– O O O D D O O O O S O–Na+ – + O N N O O S N O O Na Structure N O N O O S O O O N S O N N O O O D D O O O O O O O O O O O O DSS BS3 BS3-d4 DSG Disuccinimidyl Bis(sulfo-succinimidyl)Bis(sulfosuccinimidyl) suberate Bis(sulfosuccinimidyl)Bis(sulfo-succinimidyl) 2,2,7,7 suberate-d4 Disuccinimidyl Full name Disuccinimidyl suberate Disuccinimidyl glutarate MW 572.43 MW 576.45; Crosslink Mass 142.09 suberateMW 368.34 suberate 2,2,7,7-suberate-d4 glutarate Spacer Arm 11.4 Å Spacer Arm 11.4 Å MW 326.26 Spacer Arm 11.4 Å Spacer arm (Å) 11.4 11.4 11.4 Spacer Ar7.7m 7.7 Å Water-soluble No Yes Yes No Isotopically labeled No No Yes No MS-cleavable No No No No

2 2 Crosslinker BS G-d0 BS G-d4 DSSO DSBU

Na+O– O–Na+ Na+O– O–Na+ O O O O O O O S S A B O O O S S O O O O OO O O O O O O S O C O O S O N N O O O O N N N N NN Structure HH N N N N O O O O O O O O O O O O O O O O O DSSO (disuccinimidyl sulfoxide) O DSBU (disuccinimidyl dibutyric urea) O D D D D O Molecular weight: 388.35 Molecular weight: 426.38 Spacer arm: 10.3 Å Spacer arm: 12.5 Å BS2G-d0 DSSO Bis(sulfo-succinimidyl) Bis(sulfo-succinimidyl)BS2G-d0 Disuccinimidyl Disuccinimidyl Full name Bis(sulfosuccinimidyl) glutarate-d0 Disuccinimidyl sulfoxide MW 530.35;glutarate Crosslink Mass 96.02 Bis(sulfosuccinimidyl)2,2,4,4-suberate-d 2,2,4,4 glutarate-d4 4 sulfoxide dibutyric urea MW 388.35 Spacer Arm 7.7 Å MW 534.37; Crosslink Mass 100.04 Spacer arm (Å) 7.7 Spacer 7.7Arm 7.7 Å Spacer10.1 Arm 10.3 Å 12.5 Water-soluble Yes Yes No No Isotopically labeled No Yes No No MS-cleavable No No Yes Yes

Learn more at thermofisher.com/ms-crosslinking

32 MS-cleavable crosslinkers Features of DSSO and DSBU • Amine-reactive NHS ester (at both ends) reacts rapidly Thermo Scientific™ DSSO (disuccinimidyl sulfoxide) and with any molecule containing a primary amine DSBU (disuccinimidyl dibutyric urea, also known as BuUrBu [Note: BuUrBu stands for dibutyric urea]) are • Membrane-permeant, allowing intracellular crosslinking high-quality MS-cleavable crosslinkers that contain an • High-purity crystalline reagent can be used to create amine-reactive N-hydroxysuccinimide (NHS) ester at each high-purity conjugates end of a 7-atom and 11-atom spacer arm (Figure 42). These products are offered in convenient single-use packaging • MS-cleavable (10 x 1 mg). • Water-insoluble (dissolve first in DMF or DMSO) Chemical crosslinking in combination with mass spectrometry is a powerful method to characterize proteins. This method has been applied to recombinant and native protein complexes and, more recently, to whole- cell lysates or intact unicellular organisms in efforts to identify protein–protein interactions on a global scale.

A DSSO B DSBU

Figure 42. Spectra of BSA-crosslinked peptide identified by MS2/MS3 method and XLinkX software* using (A) DSSO and (B) DSBU crosslinkers. XLinkX software uses unique fragment patterns of MS-cleavable crosslinkers (purple annotation) to detect and filter crosslinked peptides for a database search. * Licensed from the Heck group, University of Utrecht, The Netherlands.

33 In vivo crosslinking In contrast to crosslinkers that are introduced to cells exogenously, methods exist to incorporate crosslinkers Crosslinkers are used for identification of near-neighbor into the proteome of a cell. This can be accomplished protein relationships and ligand–receptor interactions. with photoreactive crosslinkers such as Thermo Scientific™ Crosslinking stabilizes transient endogenous protein– L-Photo-Leucine (Cat. No. 22610) and L-Photo-Methionine protein complexes that may not survive traditional (Cat. No. 22615). These amino acid analogs are fed to biochemical techniques such as immunoprecipitation. The cells during cell growth and are activated with UV light most basic cellular crosslinker is formaldehyde (Thermo (Figure 43). In the experiment below, photoreactive amino Scientific™ Pierce™ 16% Formaldehyde (w/v), Methanol- acids were compared to formaldehyde treatment for free, Cat. No. 28906), which is commonly used to stabilize identifying endogenous protein complexes (Figure 44). chromatin interactions for chromatin immunoprecipitation (ChIP) assays (Thermo Scientific™ Pierce™ Agarose ChIP Kit, Cat. No. 26156). The crosslinkers chosen for these applications are usually longer than those used for subunit crosslinking. Homobifunctional, amine-reactive NHS esters or imidates and heterobifunctional, amine-reactive, photoactivatable phenyl azides are the most commonly used crosslinkers for these applications. Occasionally, a sulfhydryl- and amine-reactive crosslinker, such as Thermo Scientific™ Sulfo-SMCC (Cat. No. 22322), may be used if one of the two proteins or molecules is known to contain Figure 44. Photoreactive amino acid crosslinking and formaldehyde sulfhydryls. Both cleavable or noncleavable crosslinkers crosslinking are complementary techniques for protein interaction can be used. Because the distances between two analysis. HeLa cells were mock treated (lane 1), treated with 1% formaldehyde for 10 minutes (lane 2), or treated with Photo-Methionine molecules are not always known, the optimal length of the and Photo-Leucine followed by UV treatment (lane 3). Cells were lysed, spacer arm of the crosslinker may be determined by the and 10 µg of each was analyzed by SDS-PAGE and western blotting with use of a panel of similar crosslinkers with different lengths. antibodies against HSP90, Stat3, Ku70, and Ku86. Lower panels are β-actin (upper band) and GAPDH (lower band), which were blotted as ™ Thermo Scientific DSS (Cat. No. 21555) or its cleavable loading controls. analog DSP (Cat. No. 22585) are among the shorter crosslinkers used for protein–protein interactions.

O O O O H NHN 2 2 H N OH OH 2 H2N H H OH OH H N H L- Photo-Methionine L- Methionine N N S L- Photo-Leucine L- Leucine N

UV – + UV

Crosslinked Incubate 24 hours Harvest cells, lyse. Crosslinked at 37°C, 5% CO . 2 Run SDS-PAGE. Monomer

Add Photo-Met and Photo-Leu Expose cells to UV light Analyze by western blot dissolved in limiting media to cells. (365 nm bulb). using specific antibody.

Figure 43. In vivo crosslinking with photoreactive amino acids.

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34 Metabolic labeling

GlcNAz GalNAz ManNAz Chemoselective ligations use unique chemical functional OAc AcO groups for specific conjugation. Examples of this chemistry OAc O O N include hydrazide–aldehyde condensation, click chemistry AcO O AcO 3 AcO OAc OAc HN O 1 AcO (azide–alkyne), and Staudinger ligation (azide–phosphine). NH NH AcO N AcO OAc 3 N Click chemistry is the detection method of choice for O 3 O samples that would be compromised by direct labeling N-Azidoacetylglucosamine N-Azidoacetylgalactosamine N-Azidoacetylmannosamine or antibody-based secondary detection techniques. The click label is small enough to penetrate complex samples Extracellular Intracellular easily, and the selectivity and stability of the click reaction – OH CO2 HO OH H O provides high sensitivity and low background signal. This N O N N O HO gentle sample treatment together with the biocompatible 3 O Azido sugar Invitrogen™ Click-iT™ Plus reaction means that detection O HN O NH can be multiplexed with expressed proteins such as GFP, H H OCH H H 3 N O N S PPh protein labels such as R-PE, and a wide range of organic 2 O 3 O fluorophores. The Staudinger ligation has the best utility for Biotin-PEG3-phosphine live-cell labeling and mass spectrometry (MS) applications. New amide bond OH – The Staudinger reaction occurs between a phosphine and CO2 HO OH H O O O O N an azide to produce an aza-ylide that is trapped to form a NH Ph P HN O HO HN 2 NH O H H O stable covalent bond. Because phosphines and azides are H H S N O N absent in biological systems, there is minimal background O 3 O labeling of cells or lysates. Unlike click chemistry, Staudinger ligation requires no accessory reagents such as copper. Figure 45. Biotin labeling of azido sugars. A B Metabolic labeling involves incorporation of a chemoselective crosslinker into the proteome of living cells. This facilitates protein isolation or fluorescent labeling. One such example is the metabolic labeling of glycoproteins using azido sugars. Azido groups react with phosphines to create a stable amide bond following the Staudinger reaction. Once incorporated, azido sugars can be labeled with biotin (Figure 45) or DyLight fluorophores (Figure 46). In the examples below, U2OS cells or HK-2 cells were fed U2OS: azido-galactosamine HK-2: azido-mannosamine azido sugars, fixed, and stained with Thermo Scientific™ Figure 46. In vivo detection of metabolically incorporated azido- DyLight™ 550-Phosphine (Cat. No. 88910) or DyLight™ acetylgalactosamine using DyLight 550- and 650-Phosphine 650-Phosphine (Cat. No. 88911). labeling reagents. (A) U2OS cells were incubated with 40 µM azido- acetylgalactosamine in cell culture medium for 72 hours, and the live cells were incubated with 100 µM of DyLight 550-Phosphine. The cells were then washed, fixed with 4% paraformaldehyde, and counterstained with Hoechst™ 33342 stain (green: DyLight 550–labeled azido-galactosamine, blue: Hoechst 33342–labeled nuclei). (B) HK-2 cells were incubated with 40 µM azido-acetylmannosamine in cell culture medium for 72 hours, and the live cells were incubated with 100 µM of DyLight 650-Phosphine. The cells were then washed, fixed with 4% paraformaldehyde, and counterstained with Hoechst 33342 stain (cyan: DyLight 650–labeled azido-mannosamine, red: Hoechst 33342–labeled nuclei).

35 Cell surface crosslinking Cell membrane structural studies and biotinylation Cell membrane structural studies require reagents of varying hydrophobicity to determine the location and the Crosslinkers are often used to identify surface receptors environment within a cell’s lipid bilayer. Fluorescent tags or their ligands. Membrane-impermeant crosslinkers are used to locate proteins, lipids, or other molecules ensure cell surface–specific crosslinking. Water-insoluble inside and outside the membrane. Various crosslinkers, crosslinkers, when used in controlled amounts of reagent with differing spacer arm lengths, can be used to crosslink and reaction times, can reduce membrane penetration and proteins to associated molecules within the membrane to reaction with inner-membrane proteins. determine the distance between molecules. Successful crosslinking with shorter crosslinkers is a strong indication The sulfonyl groups attached to the succinimidyl rings of that two molecules are interacting in some manner. NHS esters result in a crosslinker that is water-soluble, Failure to obtain crosslinking with a panel of shorter membrane-impermeant, and nonreactive with inner- crosslinkers, while obtaining conjugation with the use of membrane proteins. Therefore, reaction time and quantity longer reagents, generally indicates that the molecules of crosslinker are less critical when using sulfo-NHS esters. are located in the same part of the membrane, but are Homobifunctional sulfo-NHS esters, heterobifunctional not interacting. Homobifunctional NHS esters, imidates, sulfo-NHS esters, and photoreactive phenyl azides are or heterobifunctional NHS ester/photoactivatable phenyl good choices for crosslinking proteins on the cell surface. azides are commonly used for these procedures. Although imidoester crosslinkers (imidates) are water-soluble, they Determination of whether a particular protein is located are still able to penetrate membranes. Sulfhydryl-reactive on the surface or the integral part of the membrane crosslinkers may be useful for targeting molecules with can be achieved by performing a conjugation reaction cysteines to other molecules within the membrane. of a cell membrane preparation to a known protein or radioactive label using a water-soluble or water-insoluble Thermo Scientific™ EDC (Cat. No. 22980, 22981) and crosslinker. Upon conjugation the cells may be washed, other water-soluble and -insoluble coupling reagent pairs solubilized, and characterized by SDS-polyacrylamide gel are used to study membranes and cellular structure, electrophoresis (PAGE) to determine whether the protein protein subunit structure and arrangement, enzyme– of interest was conjugated. Integral membrane proteins substrate interactions, and cell-surface and membrane will form a conjugate in the presence of a water-insoluble receptors. The hydrophilic character of EDC can result crosslinker, but not in the presence of water-soluble in much different crosslinking patterns in membrane and crosslinkers. Surface membrane proteins can conjugate subunit studies than hydrophobic carbodiimides. Often in the presence of water-soluble and water-insoluble it is best to attempt crosslinking with a water-soluble crosslinkers. The Thermo Scientific™ Pierce™ Cell Surface and water-insoluble carbodiimide to obtain a complete Protein Isolation Kit (Cat. No. 89881) is a complete set of picture of the spatial arrangements or protein–protein reagents that utilizes a cell-impermeant, cleavable biotinylation interactions involved. reagent (Sulfo-NHS-SS-Biotin) for the selective biotinylation and subsequent purification of mammalian cell surface + Cl– N NH C proteins to the exclusion of intracellular proteins. The labeled N surface proteins are affinity-purified using Thermo Scientific NeutrAvidin Agarose Resin. Figure 48. EDC can be used inED couplingC studies to study cell 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide • HCl O membrane structure. MW 191.70 NH HN Spacer Arm 0.0 Å O + – H Na O O S N O S N S S O O O O Sulfo-NHS-SS-Biotin MW 606.69 Figure 47. Sulfo-NHS-SS-BiotinSpacer isAr mused 24.3 Åfor the biotinylation and isolation of cell surface proteins.

36 Special packaging to meet specific bioconjugation needs Introduction Biocojugation reagents are highly reactive molecules and therefore may be sensitive to water, light, oxygen, or other surrounding conditions. They may form unstable intermediates that lead to undesired side products of the intended reactions. Therefore, it is critical to minimize the exposure of bioconjugation reagents to these negative environmental factors, to obtain the highest yields and quality possible. To mitigate these risks, we offer specific packaging and quality grades for your bioconjugation reagents. In addition to these options, our bioconjugation reagents are available in multiple pack sizes. Custom packaging and pack sizes are also available by request.

37 No-Weigh packaging format for bioconjugation reagents Convenient, ready-to-use vials for single- use applications

With the convenient Thermo Scientific™ No-Weigh™ packaging format, a ready-to-use solution can be made quickly and conveniently. This unique packaging format helps eliminate the need to weigh out small volumes of dry chemicals.

Once reconstituted, the reagent is ready to use at the desired concentration. Avoid weighing hassles and wasting precious reagents with our single-use Table 7. No-Weigh reagents. No-Weigh packaging format. EDC Carboxyl-to-amine DSG Highlights crosslinkers Sulfo-NHS • Helps save time—avoid weighing chemicals; just add DSP water, buffer, or solvent to create a working solution BS3 in seconds Amine-to-amine DSS crosslinkers DSSO DBSU • Helps reduce waste—small working aliquots limit the BS(PEG)5 amount of unused material discarded SMCC Amine-to-sulfhydryl Sulfo-SMCC

• Always fresh—working solution is ready to use at crosslinkers SM(PEG)2 SM(PEG) desired concentration; no need to store stock solutions 12 Photoreactive Sulfo-SANPAH The No-Weigh packaging format is available in convenient, Modification Iodoacetamide easy-to-handle screw cap vials for our most widely used reagents protein modification reagents, including reducing agents, Sulfo-NHS-LC-LC-Biotin crosslinkers, and PEG- and biotin-labeling products HPDP-Biotin Sulfo-NHS-Biotin Biotinylation (Table 7). Sulfo-NHS-SS-Biotin reagents Sulfo-NHS-LC-Biotin Sulfo-NHS-LC-Desthiobiotin Sulfo-SBED

NHS-PEG12-Biotin

NHS-PEG4-Biotin

PEGylated Maleimide-PEG2-Biotin

biotinylation reagents Hydrazide-PEG4-Desthiobiotin

Amine-PEG4-Desthiobiotin

Phosphine-PEG4-Desthiobiotin TCEP∙HCl Reducing agents DTT

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38 Premium-grade protein bioconjugation reagents Higher quality and assurance of performance from a trusted supplier

Thermo Scientific™ Pierce™ Premium Grade Reagents are the reagents of choice for applications where product integrity and risk minimization are critical (Table 8). Compared to standard grade reagents, Pierce Premium Grade Reagents provide clearly defined quality by including batch-specific information such as quality assurance review, lot sample retention, and change control notification Table 8. Pierce Premium Grade Reagents. (CCN), as well as an enhanced level of analytical testing Reagent type Premium Grade Reagent and product characterization. EDC Carboxyl-to-amine crosslinkers Sulfo-NHS Pierce Premium Grade Reagents are manufactured to the BS3 Amine-to-amine crosslinkers highest possible specifications to enable data integrity DSP Sulfo-SMCC and offer robust consistency. The consistency of each lot Amine-to-sulfhydryl crosslinkers SPDP of reagent is assessed using thorough testing procedures Sulfo-NHS-LC-Biotin Biotinylation reagents (Table 9). Sulfo-NHS-SS-Biotin Reducing agents TCEP∙HCI Highlights • Quality reagents—high-purity reagents that can be Table 9. Testing for Premium Grade Reagents.

used to create high-quality activated derivatives, labeled Specification Procedure proteins, and bioconjugates Purity Quantitative NMR using an internal standard • Product integrity—enhanced level of testing and Visual Color assessment characterization Example: sample dissolves at a specified Solubility concentration in a given solvent to yield a clear, colorless solution • Lot retention—ample supply of past lots retained to Identity Infrared (IR) spectroscopy ensure future process testing Mass identity Mass spectrometry • Change management—change control notification Water content Karl Fischer titration Inductively coupled plasma mass spectrometry (CCN) service Trace metals (ICP-MS) • Consistent manufacturing—batch-specific Elemental Reported values for C, H, N, O, and S based on analysis combustion analysis manufacturing documentation review Residual solvent Headspace gas chromatography analysis* * Performed upon request at an additional cost.

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39 Bioconjugation resources Crosslinker Selection Tool

The Crosslinker Selection Tool provides quick access to customized lists of Thermo Scientific™ Pierce™ crosslinkers that meet specific criteria, including target functional group, solubility, and the ability to permeate the cell membrane. Use the simple drop-down boxes to easily select the optimal crosslinking reagent for your application.

Learn more at thermofisher.com/crosslinking-tool

Crosslinker Application Reference Guide

Need easy access to key references? This resource can help you quickly identify relevant publications describing the use of these crosslinkers in a broad range of applications, including interaction analysis, conjugation, immobilization, in vivo crosslinking, labeling, structural analysis, and more.

Learn more at thermofisher.com/ crosslinker-publications

40 Biotinylation Reagent Selection Tool

The Biotinylation Reagent Selection Tool provides quick access to customized lists of Thermo Scientific™ biotinylation and desthiobiotin reagents that meet specific criteria, including target functional group, spacer arm length, and solubility. Use the simple dropdown boxes to easily select the optimal biotinylation reagent for your application.

Learn more at thermofisher.com/biotinylation-tool

Click-iT Labeling Technologies Guide

The Click-iT technologies guide can help you with labeling and detection of samples that would otherwise be compromised by direct labeling or antibody-based secondary detection techniques. Browse sections on Click-iT assay kits and tools to find the product that best fits your experiment.

Learn more at thermofisher.com/clickit

Fluorescence SpectraViewer Tool

The SpectraViewer is a tool to help you plan your experiments and analyses, by assisting you in choosing the best fluorophore based on your application, light sources, and filters. Easily find the right fluorophore to label your protein, antibody, or nucleic acid, over a broad range of applications and techniques.

Learn more at thermofisher.com/spectraviewer

41 Glossary of crosslinking terms

Acylation: Reaction that introduces an acyl group (–COR) Hapten: A molecule recognized by antibodies but into a compound. unable to elicit an immune response unless attached to a carrier protein. Haptens are usually, but not always, small Aryl azide: Compound containing a photoreactive (<5 kDa) molecules. functional group (e.g., phenyl azide) that reacts nonspecifically with target molecules. Homobifunctional crosslinker: Reagent with two identical reactive groups used to link two Carbodiimide: Reagent that catalyzes the formation of molecules or moieties. an amide linkage between a carboxyl (–COOH) group and a primary amine (–NH2) or a hydrazide (–NHNH2). These Heterobifunctional crosslinker: Reagent with reagents do not result in the formation of a cross-bridge two different reactive groups used to link two and have been termed zero-length crosslinkers. molecules or moieties.

Crosslinker: A reagent that will react with functional Hydrophilic: Substances that readily dissolve in water. groups on two or more molecules to form a covalent linkage between the molecules. Hydrophobic: Substances with limited solubility in water.

Conjugation reagent: A crosslinker or other reagent for N-Hydroxysuccinimidyl (NHS) ester: Acylating reagents covalently linking two molecules. commonly used for crosslinking or modifying proteins.

They are specific for primary (–NH2) amines between Diazirine crosslinker: The succinimidyl-ester diazirine pH 7 and 9, but are generally the most effective at neutral (SDA) crosslinkers combine amine-reactive chemistry pH. These esters are subject to hydrolysis, with half- with an efficient diazirine-based photochemistry for lives approximating 1–2 hours at room temperature at photocrosslinking to nearly any other functional group. neutral pH. The photoactivation of diazirine with long-wave UV light (330–370 nm) creates carbene intermediates. These Imidate crosslinker: Primary amine-reactive functional intermediates can form covalent bonds via addition group that forms an amidine bond. The ε-amine in lysine reactions with any amino acid side chain or peptide and N-terminal amines are the targets in proteins. Imidates backbone at distances corresponding to the spacer react with amines in alkaline pH conditions (pH range 7.5– arm lengths. 10) and hydrolyze quickly, with half-lives typically around 10–15 minutes at room temperature and pH 7–9. At pH >11, Disulfide bonds: Oxidized form of sulfhydryls (–S–S–); the amidine bond is unstable, and crosslinking can be formed in proteins through –SH groups from two cysteine reversed. The amidine bond is protonated at physiological molecules. These bonds often link polypeptide chains pH; therefore, it carries a positive charge. together within the protein and contribute to a protein’s tertiary structure. Imidoester: Amine-reactive functional group of an imidate crosslinker. α-Haloacyl: Functional group (e.g., iodoacetyl) that targets nucleophiles, especially thiols. α-Haloacyl compounds Immunogen: A substance capable of eliciting an have a halogen atom such as iodine, chlorine, or bromine immune response. attached to an acyl group on the molecule. These alkylating reagents degrade when exposed to direct light Integral membrane protein: Protein that extends or reducing agents, resulting in loss of the halogen and the through the cell membrane and is stabilized by appearance of a characteristic color. hydrophobic interactions within the lipid bilayer of the membrane.

42 Ligand: A molecule that binds specifically to another Polymer: A molecule composed of many molecule. For example, a protein that binds to a receptor. repeating monomers.

Moiety: An indefinite part of a sample or molecule. Pyridyl disulfide: Aromatic moiety with a disulfide attached to one of the carbons adjacent to the Monomer: A single unit of a molecule. nitrogen in a pyridine ring. Pyridine 2-thione is released when this reagent reacts with a NHS: Abbreviation for N-hydroxysuccinimide. sulfhydryl (–SH)-containing compound.

Nitrene: Triple-bonded nitrogen-to-nitrogen reactive group Spacer arm: The part of a crosslinker that is incorporated formed after exposure of an azido group to UV light. Its between two crosslinked molecules and serves as a bridge reactivity is nonspecific and short lived. between the molecules.

Nonselective crosslinking: Crosslinking using a reactive Substrate: A substance upon which an enzyme acts. group, such as nitrenes or aryl azides, which react so quickly and broadly that specific groups are not easily and Sulfhydryl: –SH groups present on cysteine efficiently targeted. Yields are generally low, with many residues in proteins. different crosslinked products formed. Thiols: Also known as mercaptans, thiolanes, sulfhydryls, Nonspecific crosslinking: Another term for nonselective or –SH groups, these are good nucleophiles that may be crosslinking. targeted for crosslinking.

Oligomer: A molecule composed of several monomers. Ultraviolet: Electromagnetic radiation of wavelengths between 10 and 390 nm. Photoreactive: A functional group that becomes reactive upon excitation with light at a particular range of wavelengths.

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SAT(PEG)4 404. Hermanson GT (2008) Bioconjugate Techniques (2nd ed.). Academic Press.

49 Ordering information Homobifunctional crosslinkers

Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Amine-to-amine reactive (NHS ester)

O O DSG (disuccinimidyl glutarate) O O 50 mg 20593 326.26 7.7 N N O O Pierce DSG, No-Weigh Format O O 10 x 1 mg A35392 DSG Disuccinimidyl glutarate O 50 mg 21655 DSS (disuccinimidyl suberate) O MW 326.26 O OSpacer Arm 7.7 Å N 1 g 21555 368.35 11.4 N O O O Pierce DSS, No-Weigh Format O 10 x 2 mg A39267 DSS 50 mg 21580 Disuccinimidyl suberate BS3 (bis(sulfosuccinimidyl)suberate) OMW 368.34 Na+O– 1 g 21586 SpacerO Arm 11.4Å O O S O–Na+ 572.43 11.4 N O 100 mg PG82083 Pierce™ Premium Grade BS3 O O N S O O O O 1 g PG82084 O Pierce BS3, No-Weigh Format BS3 10 x 2 mg A39266 Bis(sulfosuccinimidyl) suberate BS(PEG) (PEGylated MW 572.43 5 O O 100 mg 21581 bis(sulfosuccinimidyl)suberate) O Spacer Arm 11.4Å O 532.50 21.7 N O O N O O O O O O O Pierce BS(PEG)5, No-Weigh Format 10 x 1 mg A35396 BS(PEG)5 O Bis(succinimidyl) penta(ethylene glycol) O BS(PEG) (PEGylated O MW 532.50 O 9 708.71 35.8 N O O O O N 100 mg 21582 bis(sulfosuccinimidyl)suberate) O O SOpacer Arm 21.7OÅ O O O O O

BS(PEG)9 Bis(succinimidyl) nona(ethylene glycol) 1 g 22585 DSP (dithiobis(succinimidyl MW 708.O71 propionate)), Lomant’s Reagent O SpacerO Arm 35.8Å 50 mg 22586 O S N 404.42 12.0 N S O 1 g PG82081 Pierce Premium Grade DSP O O 10 g PG82082 O Pierce DSP, No-Weigh Format 10 x 1 mg A35393 O DSP Na+O– Dithiobis(succinimidylprO opionate) O DTSSP (3,3´-dithiobis(sulfosuccinimidyl O S N MW 404.42 S O O 608.51 12.0 O S N 50 mg 21578 propionate)) O Spacer Arm 12.0 Å S O O O O–Na+ O

O DTSSP 3,3'O -Dithiobis(sulfOH osuccinimidylprO opionate) N O MW 608.51 DST (disuccinimidyl tartrate) 344.24 6.4 O N 50 mg 20589 Spacer Arm 12.0 Å O OH O O DST O O Disuccinimidyl tartraOte O BSOCOES (bis(2-(succinimido- O O 436.35 13.0 N MW 344.23S N 50 mg 21600 oxycarbonyloxy)ethyl)sulfone) OO SpacerO Arm 6.O4 Å O O

BSOCOES O OBis[2-(succinimidooxOycarbonyloxy)ethyl]sulfoneO EGS (ethylene glycol bis(succinimidyl O MW 436.35 O N 456.36 16.1 N O O 1 g 21565 succinate)) Spacer Arm 13.0 Å O O O O EGS Ethylene glycol bis(succinimidylsuccinate) MW 456.36 50 Spacer Arm 16.1 Å Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

O Na+O– O O O Sulfo-EGS (ethylene glycol O 660.45 16.1 O S N O O 50 mg 21566 bis(sulfosuccinimidyl succinate)) O O O N S O O O O O–Na+ O

O Sulfo-EGS EthyN lene glycol bis(sulfosuccinimidylsuccinate) O O MW 660.45 O Spacer Arm 16.1 Å TSAT (tris-(succinimidyl) 482.36 4.2 50 mg 33063 aminotriacetate) N O O O O N O O N O O Amine-to-amine reactive (NHS ester), deuterated or MS-cleavableTSAT Tris-succinimidyl aminotriacetate Na+O– MW 482.36 O–Na+ SOpacer Arm 4.2 Å O S S BS2G-d (bis(sulfosuccinimidyl) O O O O 0 530.35 7.7 O O 10 mg 21610 glutarate-d ) 0 N N O O O O

Na+O– BS2G-d0 O–Na+ O O S Bis(sulfosuccinimidyl) glutarate-dS0 2 O MW 53O 0.35; Crosslink MassO 96.02 O BS G-d4 (bis(sulfosuccinimidyl) O O 534.38 7.7 Spacer Arm 7.7 Å 10 mg 21615 2,2,4,4-glutarate-d4) N N O O O D D D D O

O BS2G-d0 Na+O– Bis(sulfosuccinimidyl)O 2,2,4,4 glutarate-d4 O BS3-d (bis(sulfosuccinimidyl) O S O–Na+ 0 572.43 11.4 MW 534.N37; Crosslink Mass 100.04 O 10 mg 21590 suberate-d ) O SOpacer Arm 7.7 Å N S O 0 O O O O

O BS3-d0 Na+O– Bis(sulfO osuccinimidyl) DsuberaD te-d0 O BS3-d (bis(sulfosuccinimidyl) O S O–Na+ 4 576.45 11.4 NMW 576.45; Crosslink Mass 13O8.07 10 mg 21595 2,2,7,7-suberate-d ) O O Spacer Arm 11.4 Å N S O 4 O D D O O O BS3-d O O 4 O Bis(sulfosuccinimidyl) 2,2,7,7 suberate-d4 O S O DSSO (disuccinimidyl sulfoxide) 388.35 10.1 N MW 576.45; Crosslink MassN 142.09 10 x 1 mg A33545 O Spacer ArOm 11.4 Å O O

DSSOO A O O O B O O O DisuccinimidylC sulfoxide O O S O N N N N NN DSBU (disuccinimidyl dibutyric urea) 426.38 12.5 MW 38HH8.35 10 x 1 mg A35459 O O O O Spacer Arm 10.3 Å O O O O DSSO (disuccinimidyl sulfoxide) DSBU (disuccinimidyl dibutyric urea) Molecular weight: 388.35 Molecular weight: 426.38 Spacer arm: 10.3 Å Spacer arm: 12.5 Å

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51 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Amine-to-amine reactive (imidoester or difluoro) + – NH2 Cl O DMA (dimethyl adipimidate) 245.15 8.6 O 1 g 20660 + – NH2 Cl DMA + – + – 50 mg 21666 DimeNHthyl2 adipimidCl ate • 2HNHCl2 Cl DMP (dimethyl pimelimidate) 259.17 9.2 MW 245.15 O O 1 g 21667 Spacer Arm 8.6 Å NH +Cl– 2 DMP Dimethyl pimelimidate • 2HClO DMS (dimethyl suberimidate) 273.20 11.0 O 1 g 20700 MW 259.17 NH +Cl– Spacer arm 9.2 Å 2 DMS NH +Cl– Dimethyl suberimidate • 2HCl2 O S DTBP (Wang and Richard’s Reagent) 309.28 11.9 MW 27S3.20 O 1 g 20665 Spacer Arm 11.0 Å + – NH2 Cl

FFDTBP Dimethyl 3,3' dithiobispropionimidate • 2HCl DFDNB (1,5-difluoro-2,4- O 204.09 3.0 –O MW 309.28 50 mg 21525 dinitrobenzene) N+ N+ Spacer Arm 11.9Å O O– Sulfhydryl-to-sulfhydryl reactive (maleimide) DFDNB 1,5-difluoro-2,4-dinitrobenzene MW 204.09O OSpacer arm 3.0 Å N BMOE (bismaleimidoethane) 220.18 8.0 N 50 mg 22323 O O

BMOE O BismaleimidoO ethane MW 220.18 N BMB (1,4-bismaleimidobutane) 248.23 10.9 N 50 mg 22331 Spacer Arm 8.0 Å O O

BMB O O1,4-Bismaleimidobutane MW 248.23 N BMH (bismaleimidohexane) 276.29 16.1 N 50 mg 22330 Spacer Arm 10.9 Å O O

BMH O O Bismaleimidohexane BM(PEG)2 (1,8-bismaleimido- MWO 276.29 N 308.29 14.7 N O 50 mg 22336 diethyleneglycol) Spacer Arm 13.0 Å O O

O BM(PEG)2 O BM(PEG) (1,11-bismaleimido- 1,8-BismaleimidodieO thyleneglycoOl 3 352.34 17.8 N O N 50 mg 22337 triethyleneglycol) MW 308.29 Spacer Arm 14.7 Å O O

BM(OPEG)3 O 1,11-Bismaleimidotriethyleneglycol S N DTME (dithiobismaleimidoethane) 312.37 13.3 N S MW 352.34 50 mg 22335 Spacer Arm 17O.8 Å O

DTME DithiobismaleimidoethaneO N O MW 312.36 O Spacer Arm 13.3 Å TMEA (tris(2-maleimidoethyl)amine) 386.36 10.3 N 50 mg 33043 N O

O N

O

TMEA Tris-(2-maleimidoethyl)amine MW 386.36 Spacer Arm 10.3 Å 52 Heterobifunctional crosslinkers

Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Amine-to-sulfhydryl reactive (NHS-haloacetyl)

O O SIA (succinimidyl iodoacetate) 283.02 1.5 N I 50 mg 22349 O O

O SIA H SBAP (succinimidyl 3-(bromo- SuccinimidylO iodoacetateN 3 07.10 6.2 N Br 50 mg 22339 acetamido)propionate) MW 283.02 Spacer ArmO 1.5 Å O O

SBAP H N Succinimidyl-3-(bromoacetamidoO )propionatI e SIAB (succinimidyl (4-iodoacetyl) 402.14 10.6 O MW 307.10 O 50 mg 22329 aminobenzoate) N Spacer Arm 6.2 Å O O

SIAB H N Succinimidyl (4O-iodoacetyl) aminobenzoate I Sulfo-SIAB (sulfosuccinimidyl + – MW 402.14 504.19 10.6 Na O O O 50 mg 22327 (4-iodoacetyl)aminobenzoate) O S SpacerN Arm 10.6 Å O O O

Amine-to-sulfhydryl reactive (NHS-maleimide) Sulfo-SIAB Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate O O O MW 504.19 AMAS (N-α-maleimidoacet- Spacer Arm 10.6 Å 252.18 4.4 N N 50 mg 22295 oxysuccinimide ester) O O O

AMAS O O M.W. 252.18 BMPS (N-β-maleimidopropyl- 266.21 5.9 SpacerO Arm 4.4N Å 50 mg 22298 oxysuccinimide ester) N O O O

O BMPS O GMBS (N-γ-maleimidobutyryl- N-(ß-MaleimidoprO opyloxy) succinimide ester 280.23 7.3 NN 50 mg 22309 oxysuccinimide ester) MW 266.21 SpacerO Arm 5.9 Å O O

O GMBS O Na+O– Sulfo-GMBS (N-γ-maleimidobutyryl- N-(γ-Maleimidobutyryloxy)succinimideO ester 382.28 7.3 O S N N 50 mg 22324 oxysulfosuccinimide ester) MW 280.23 O O Spacer ArmO 7.3 Å O

O Sulfo-GMBS O N-(у-Maleimidobutyryloxy) sulfosuccinimide ester MBS (m-maleimidobenzoyl- O 314.25 7.3 N MW 382.28N 50 mg 22311 N-hydroxysuccinimide ester) SOpacer Arm 7.3 Å O O

MBS O O m-Maleimidoben+ – zoyl-N-hydroxysuccinimide ester Sulfo-MBS (m-maleimidobenzoyl- Na O O 416.30 7.3 MWN 314.25 N 50 mg 22312 N-hydroxysulfosuccinimide ester) O S Spacer Arm 7.3 Å O O O O

Sulfo-MBS SMCC (succinimidyl 4-(N-maleimido- O m-Maleimidobenzoyl-N-hydroxysulfosuccinimideO ester 50 mg 22360 methyl)cyclohexane-1-carboxylate) O N MW 416.29 334.32 8.3 N SOpacer Arm 7.3 Å O Pierce SMCC, No-Weigh Format O 10 x 1 mg A35394

(1r,4r)-2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate

Chemical Formula:C16H18N2O6 Exact Mass: 334.12 53 Molecular Weight: 334.32 m/z: 334.12 (100.0%), 335.12 (17.7%), 336.12 (2.8%) Elemental Analysis: C, 57.48; H, 5.43; N, 8.38; O, 28.71 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Sulfo-SMCC (sulfosuccinimidyl 50 mg 22322 4-(N-maleimidomethyl)cyclohexane- O 1-carboxylate) 1 g 22122 O N + – 436.37 8.3 Na O O 100 mg PG82085 Pierce Premium Grade Sulfo-SMCC O S N O 1 g PG82086 O O Pierce Sulfo-SMCC, No-Weigh O 10 x 2 mg A39268 Format Sulfo-SMCC SulfosuccinimidylO 4-(N-maleimidomethyl)cyclohexane-1-carboxylate MW 436.37 OH Pierce EMCA (N-ε-maleimidocaproic N 211.21 9.4 Spacer Arm 8.3Å 1 g 22306 acid) O O

O EMCA O M.W. 211.21 EMCS (N-ε-maleimidocaproyl- O 308.29 9.4 N Spacer Arm 9.4 Å N 50 mg 22308 oxysuccinimide ester) O O O

O EMCS O Na+O– Sulfo-EMCS (N-ε-maleimidocaproyl- N-(ε-MaleimidocaproyOloxy) succinimide ester 410.33 9.4 N N 50 mg 22307 oxysulfosuccinimide ester) O S MW 308.29 O Spacer ArmO 9.4 Å O O

O Sulfo-EMCS N-(ε-MaleimidocaprO oyloxy) sulfosuccinimide ester N O SMPB (succinimidyl MW 410.33 356.33 11.6 50 mg 22416 O Spacer Arm 9.4 Å 4-(p-maleimidophenyl)butyrate) O N

O

O SMPB Na+O– Succinimidyl 4-(p-maleimidopheO nyl)butyrate N O Sulfo-SMPB (sulfosuccinimidyl O S MW 356.33 458.38 11.6 O O 50 mg 22317 4-(N-maleimidophenyl)butyrate) SpacerO Arm 11.6 Å N

O

O Sulfo-SMPBO O SMPH (succinimidyl 6-((β-male- O 379.36 14.2 SulfosuccinimidylN 4-(p-maleimidopheN nyl)buN tyrate 50 mg 22363 imidopropionamido)hexanoate)) MW 458.3H7 O O Spacer Arm 11.6 Å O

O SMPH O SuccinimidylO 6-[(β-maleimidopropionamido)hexanoatOe] LC-SMCC (succinimidyl 4-(N-male- N N imidomethyl)cyclohexane-1-carboxy- 4 47.48 16.2 MW 379.36 H 50 mg 22362 O Spacer Arm 14.2 Å N (6-amidocaproate)) O O n = 4 SM(PLC-SMCEG)nC O SM(PEG)4 Succinimidyl 4-(N-maleimidometO NHS-PhyEGl)cyn-Malcloheeimidexane-1-carboxy-(6-amidocaproate) Sulfo-KMUS (N-κ-maleimido- Na+O– MW 513.30 Succinimidyl-([N-maleimidoprO MWopionamid 447.48o]-#ethyleneglycol) esNter undecanoyl-oxysulfosuccinimide 480.46 16.3 N Spacer arm 24.6 Å50 mg 21111 O S Spacer Arm 16.2 Å ester) O O O O n = 6 Spacer arm SM(PEG)6 Sulfo-KMUS SM(PEG) (PEGylated SMCC O O MW 601.60 100 mg 22102 2 N-(k-Maleimidoundecanoyloxy) sulfosuccinimide ester crosslinker) O Spacer arm 32.5 Å MW 480.46H 1 g 22103 425.39 17.6 N N N O SpacerO Arm 16.3 Å Pierce SM(PEG) , No-Weigh Format n = 8 10 x 1 mg A35397 2 O 2 O O SM(PEG)8 O MW 689.71 O 100 mg 22104 H Spacer arm 39.2 Å SM(PEG)4 (PEGylated SMCC O O O N N 513.5 24.6 N O O crosslinker) O n = 12 O O O O 1 g 22107 O H SM(PEG)12 O O N N MW 865.92 N O SM(PEG)4 Spacer arm 53.4 Å O C22H31N3OO11 O Mol. Wt.: 513.50 O n = 24 SM(PEG) SM(PEG) 54 2 24 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihMWydro 425.-1H-39pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoMWxa-4-a 1394zanonadecan-.55 19-oate Spacer arm 17.6 Å Spacer arm 95.2 Å Chemical Formula:C22H31N3O11 Exact Mass: 513.20 Molecular Weight: 513.50 m/z: 513.20 (100.0%), 514.20 (24.6%), 515.20 (5.3%), 514.19 (1.1%) Elemental Analysis: C, 51.46; H, 6.08; N, 8.18; O, 34.27 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

O O SM(PEG) (PEGylated, long-chain H 6 601.6 32.5 O O O O N N 100 mg 22105 SMCC crosslinker) N O O O O O O O SM(PEG)6

M.W. 601.60 O O Spacer Arm 32.5Å H SM(PEG)8 (PEGylated, long-chain O O O O O N N 689.71 39.2 N O O O O 100 mg 22108 SMCC crosslinker) O O O 2,5-dioxopyrrolidin-1-yl 1-O(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22-hexaoxa-4-azapentacosan-25-oate

Chemical Formula: C26H39N3O13 SM(PEG)8 SM(PEG) (PEGylated, long-chain Exact Mass: 601.25 O 100 mg 22112 12 O C30H47N3O15 H O O O MolecularO Weight: O601.60 O O N N SMCC crosslinker) N O O Mol. WtO.: 689.71 O O O 865.92 53.4 m/z: 601.25 (100.0%), 602.25 (30.2%), 603.25 (6.8%), 604.26 (1.1%) 1 g 22113 O O O O Elemental Analysis: C, 51.91; H, 6.53; N, 6.98; O, 34.57 Pierce SM(PEG)12, No-Weigh Format 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxoSM(PEG)12-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-o10ate x 1 mg A35398 Chemical FormulC38H63N3O19a:C H N O Mol. Wt.:30 865.9247 3 15 O Exact Mass:O 689.30 O MolecularH Weight: 689.71 SM(PEG)24 (PEGylated, long-chain 1394.55 95.2 2,5-dioxopyrrolidin-1-ylmN/z: 689.30 (1 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4-azatrite00.0%), 690.30 N(34.1%), 691.31N (5.5%), 691.30 (3.5%), 692.31 (1.6%) tracontan-43-oate102 mg 22114 SMCC crosslinker) O ElemenO tal Analysis: C, 52.24; H, 6.87; N, 6.09; O, 34.80 Chemical Formula: C38H63N3O19 O 24 O ExactO Mass: 865.41 Molecular Weight: 865.92 m/z: 865.41 (100.0%), 866.41 (42.5%), 867.41 (12.9%), 868.41 (1.8%), 868.42 (1.2%), 866.40 (1.1%) Amine-to-sulfhydryl reactive (NHS-pyridyldithiol) Elemental Analysis: C, 52.71; H, 7.33; N, 4.85; O, 35.11 SPDP (succinimidyl 3-(2-pyridyldithio) O 50 mg 21857 propionate) O S 312.37 6.8 N S N 100 mg PG82087 Pierce™ Premium Grade SPDP O O 1 g PG82088

O SPDP O LC-SPDP (succinimidyl SuccinimidylO 3-(2-pyridyldithio)propionate S N 6-(3(2-pyridyldithio)propionamido) 425.52 15.7 N MW 312.36 N S 50 mg 21651 H hexanoate) SpacerO Arm 6.8 Å O

O LC-SPDP O + – Sulfo-LC-SPDP (sulfosuccinimidyl SuccinimidylNa O 6-[3O(2-pyridyldithio)propionamido]hexanoatSe N N N S 6-(3´-(2-pyridyldithio)propionamido) 527.57 15.7 O S MW 425.52 H 50 mg 21650 hexanoate) O SOpacer Arm 15.7 Å O Sulfo-LC-SPDP SulfosuccinimidylO 6-[3'-(2-pyridylditN hio)propionamido]hexanoate SMPT (4-succinimidyloxycarbonyl- MW 527.57 388.46 20.0 50 mg 21558 α-methyl-α(2-pyridyldithio)toluene) N O SpacerSS Arm 15.7 Å

O O SMPT SuccinimidylO oxycarbonyl-methyl-(2-pyridyldithio)toluene N H PEG -SPDP (PEGylated, long-chain OOMW 388.46 O N S 4 559.17 25.7 N O O S 100 mg 26128 SPDP crosslinker) Spacer Arm 11.2 Å O O O

O PEG4-SPDPO O O S PEG -SPDP (PEGylated, long-chain 2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimideN 12 912.07 54.1 N S 100 mg 26129 12 MWH 559.17 SPDP crosslinker) O N O Spacer arm 25.7 Å

Carboxyl-to-amine reactive (carbodiimide plus NHS ester) PEG12-SPDP 2-Pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide MW 912.07 10 mg 77149 EDC (1-ethyl-3-(3-dimethylamino- Spacer arm 54.1 Å 5 g 22980 propyl)carbodiimide hydrochloride) + Cl– 25 g 22981 N NH 191.70 0 C 1 g PG82079 N Pierce Premium Grade EDC 25 g PG82073 EDC 500 g PG82074 Pierce EDC, No-Weigh Format 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide • HCl 10 x 1 mg A35391 MW 191.70 Spacer Arm 0.0 Å

55 NR CS O O Isothiocyanate Cl R S Cl R N NH C NR CO O H N Carboxylic PrimarIsocyanatey amine Sulfonyl Ochloride StableAldehyde conjugate Carbodiimide acid 2 NH (amide bond) O O 2 O N O – 1 H F Ordering Information (continued) Cl 2 O R R′ OO H RN R OH N+ 1 Spacer N R O Carbonate Acyl azide AnhydrideO Fluorobenzene Product MW arm (Å) Structure Hydrolysis F Quantity Cat. No. H O O 2 OH F F O N 1 O O NHS (N-hydroxysuccinimide) 115.09 NA N NH2 O 25 mg 24500 – O NH O R O F Cl 1 HO R CH S R– +NH O 3 O O O F O-acylisourea NHS ester Imidoester Epoxide Fluorophenyl ester N 500 mg 24510 Sulfo-NHS intermediate O O 1 NH2 (N-hydroxysulfosuccinimide) N O O (unstable) S O– 2 5 g 24525 C O N Amine-reactive EDC 217.13 NA 500 mg PG82071 Pierce Premium Grade Sulfo-NHS sulfo-NHS ester Primary crosslinker N HO (dry-stable) amine 10 g PG82072 O Pierce Sulfo-NHS, No-Weigh Format 10 x 2 mg A39269 Sulfo-NHS

Sulfhydryl-to-carbohydrate or -carboxyl (maleimide, pyridyldithiol/hydrazide, or isocyanate)

O O H BMPH (N-β-maleimidopropionic acid N N 297.19 8.1 NH + –O CF 50 mg 22297 hydrazide) 3 3 O O

BMPH O O N-ß-Maleimidopropionic acidO hydrazide • TFA EMCH (N-ε-maleimidocaproic acid MW 297.19 225.24 11.8 N NH + –O CF 50 mg 22106 hydrazide) Spacer Arm 8.1 Å N 3 3 H O

EMCH H N + – N-O ε-Maleimidocaproic acid hyNHdraz3 Clide • TFA MPBH (4-(4-N-maleimidophenyl) MW 339.O27 309.75 17.9 N 50 mg 22305 butyric acid hydrazide) Spacer Arm 11.8 Å O

MPBH O 4-(4-N-Maleimidophenyl)butyric acid hydrazide • HCl O KMUH (N-κ-maleimidoundecanoic MW 309.75 H 295.38 19.0 N N 50 mg 22111 acid hydrazide) Spacer Arm 17.9 Å + – NH3 O CF3 O O

N KMUH PDPH (3-(2-pyridyldithio)propionyl N-κ-MaleimidoundecanoicH acid hydrazide • TFA 229.32 9.2 S N 50 mg 22301 hydrazide) S MWNH 402 9.40 SOpacer Arm 19.0 Å

PDPHN O C 3-(2-Pyridyldithio)propionyl Ohydrazide PMPI (p-maleimidophenyl isocyanate) 214.18 8.7 N MW 229.32 50 mg 28100 Spacer Arm 9.2 Å O

Photoreactive (NHS ester and aryl azide, phenyl azide, diazirine,PM orPI psoralen) N-(p-Maleimidophenyl)isocyanate O MWO 214.18 SNpacer Arm 8.7 ÅN ANB-NOS (N-5-azido-2- O N+ 305.20 7.7 N– 50 mg 21451 nitrobenzoyloxysuccinimide) O –O N+ O ANB-NOS Sulfo-SANPAH (sulfosuccinimidyl O N-5-Azido-2-nitrobenzoyloxysuccinimide 6-(4´-azido-2´-nitrophenylamino) N+ N 50 mg 22589 MW 305.20 – + hexanoate) O O N – Na+O–Spacer Arm 7.7 Å N 492.40 18.2 O O S N N Pierce Sulfo-SANPAH, H O O 10 x 1 mg A35395 No-Weigh Format O Sulfo-SANPAH Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate MW 492.40 Spacer Arm 18.2 Å

56 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

O N N SDA (NHS-Diazirine) (succinimidyl O 225.20 3.9 N 50 mg 26167 4,4´-azipentanoate) O O

O NN + – Sulfo-SDA (Sulfo-NHS-Diazirine) Na O O 327.25 3.9 N 50 mg 26173 (sulfosuccinimidyl 4,4´-azipentanoate) O S O O O

O Sulfo-SDA (Sulfo-NHS-Diazirine) LC-SDA (NHS-LC-Diazirine) O NN MW 327.25 H (succinimidyl 6-(4,4´-azipentanamido) 338.36 12.5 N Spacer arm 3.9 Å N 50 mg 26168 hexanoate) O O O LC-SDA NHS-LC-Diazirine Na+O– Spacer Arm 12.5 Å O S Sulfo-LC-SDA (Sulfo-NHS-LC- OO Diazirine) (sulfosuccinimidyl 440.40 12.5 O NN 50 mg 26174 H 6-(4,4´-azipentanamido)hexanoate) N N O O O

Sulfo-LC-SDA O (Sulfo-NHS-LC-Diazirine) NN SDAD (NHS-SS-Diazirine) H O MWS 440.40 N (succinimidyl 2-((4,4´-azipentanamido) 388.46 13.5 N Spacer armS 12.5 Å 50 mg 26169 ethyl)-1,3´-dithiopropionate) O O O

SDAD NN Sulfo-SDAD (Sulfo-NHS-SS- O (NHS-SS-Diazirine) Na+O– H Diazirine) (sulfosuccinimidyl OMW 388.46S N N S 490.51 13.5 O S Spacer arm 13.5 Å 50 mg 26175 2-((4,4´-azipentanamido)ethyl)- O O O 1,3´-dithiopropionate) O

Sulfo-SDAD (Sulfo-NHS-SS-Diazirine) MWO 490.51 O Spacer arm 13.5 Å SPB (succinimidyl-(4-(psoralen- O 385.32 8.6 N O 50 mg 23013 8-yloxy))-butyrate) O O O O

SPB

SuccinimidylN-[–4-(psoralen-8-yloxy)]-butyrate N+ MW 385.32 O SNpacer Arm 8.5-9.5 Å Sulfo-SBED Biotin Label 19.1 Å NH HN 10 mg 33033 Transfer Reagent O H HN N S 9.1 Å O 19.1 HN O 879.97 13.7 9.1 S 13.7 Å S

Pierce Sulfo-SBED Biotin Label O O Sulfo-SBED MW 879.98 10 x 1 mg A39260 Transfer Reagent, No-Weigh Format O N O

O S O Na+O–

Chemoselective ligation (NHS ester and azide-phosphine or -alkyne)

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57 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. O O NHS-Azide 198.14 2.5 N N 10 mg 88902 O 3 O

ONHS-Azide MW 198.14 O O O N3 SpacerN Arm 2.5 Å O O NHS-PEG4-Azide 388.37 18.9 100 mg 26130 O O

NHS-PEG4-Azide O MW 388.37 Spacer Arm 18.9 Å O O O NHS-Phosphine 461.40 5.4 N P 10 mg 88900 O O

NHS-Phosphine O Alkyne, Succinimidyl Ester MW 461.40O Spacer Arm 5.4 Å (3-propargyloxypropanoic acid, 225.20 7.7 CH CCH2OCH2CH2 C O N 1 mg A10279 succinimidyl ester) O

O Iodoacetamide Alkyne 223.01 7.8 1 mg I10189 ICH2 C NHCH2C CH

O – + Click-iT AHA (L-azidohomoalanine) 258.16 NA NN NCH2CH2 CH OHC 5 mg C10102

• NH2 CF3COOH

O

Click-iT HPG (L-homopropargyl- CH CCH CH CH C OH 127.14 NA 2 2 5 mg C10186 glycine)

NH2 Chemoselective ligation

OAc AcO OAc O O O AcO N GlcNAz (N-azidoacetylglucosamine, HN 3 430.37 NA AcO OAc AcO OAc 5 mg 88903 tetraacylated) NH NH AcO O N AcO 3 N OAc O 3 O AcO

OAc AcOAc4GlcNAzOAc Ac4GalNAzO Ac4ManNAz O N-Azidoacetyl-O N-Azidoacetyl- N-Azidoacetyl- AcO N GalNAz (N-azidoacetylgalactosamine, glucosamine, galactosamine,HN 3 mannosamine, 430.37AcO NAOAc AcO OAc 5 mg 88905 tetraacylated) NH tetraacylatedNH AcO tetraacylatedO tetraacylated N AcO 3 N OAc O 3 O AcO

OAc AcOAc4GlcNAzOAc Ac4GalNAzO Ac4ManNAz O O ManNAz AcO N-Azidoacetyl- N-Azidoacetyl- N N-Azidoacetyl- glucosamine, galactosamine,HN 3 mannosamine, AcO OAc AcO OAc (N-azidoacetylmannosamine, NH 430.37 tetraacylatedNHNA AcOtetraacylatedO tetraacylated 5 mg 88904 N AcO tetraacylated) 3 N OAc O 3 O AcO

Ac4GlcNAz Ac4GalNAz Ac4ManNAz Photoreactive amino acidsN-Azidoacetyl- N-Azidoacetyl- N-Azidoacetyl- glucosamine, galactosamine, mannosamine, tetraacylated tetraacylated tetraacylatedO H N 2 OH L-Photo-Leucine 143.15 0 H N 100 mg 22610 N

Photo-L-LeucineO H N MW2 143.14OH L-Photo-Methionine 157.17 0 H 100 mg 22615

N N

Photo-L-Methionine MW 157.17 58 Biotin and desthiobiotin labeling reagents

Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Amine-reactive

O

NH HN EZ-Link NHS-Biotin 341.38 13.5 O 100 mg 20217 O N S O O NHS-Biotin O MW 341.38 H N SpacerO Arm 13.5 Å O EZ-Link NHS-Desthiobiotin 311.33 9.7 N 50 mg 16129 O N H O NHS-Desthiobiotin O MW 311.33 EZ-Link Sulfo-NHS-Biotin Spacer Arm 9.7 Å NH 50 mg 21217 HN O 443.43 13.5 + – Na O O S EZ-Link Sulfo-NHS-Biotin, O S N 10 x 1 mg A39256 No-Weigh Format O O O

Sulfo-NHS-Biotin O MW 443.43 Spacer Arm 13.5 Å NH O HN EZ-Link NHS-LC-Biotin 454.54 22.4 O 50 mg 21336 H N N O S O O

NHS-LC-Biotin H N O O + – MW 454.54 O EZ-Link Sulfo-NHS-LC-Desthiobiotin, Na O Spacer Arm 22.4 Å O N 526.54 17.3 O S N N 5 x 1 mg A39265 No-Weigh Format H H O O O

EZ-Link Sulfo-NHS-LC-Biotin Sulfo-NHS-LC-Desthiobiotin 100 mg 21335 MW 526.54 Spacer Arm 17.3 Å O 100 mg PG82075 NH Pierce Premium Grade O HN Na+ O– O Sulfo-NHS-LC-Biotin 556.59 22.4 H O S N N 1 g PG82076 S O O O O EZ-Link Sulfo-NHS-LC-Biotin, 10 x 1 mg A39257 No-Weigh Format Sulfo-NHS-LC-Biotin MW 556.59 Spacer Arm 22.4 Å O

NH HN O O EZ-Link NHS-LC-LC-Biotin 567.70 30.5 H 50 mg 21343 O N N N S H O O O NHS-LC-LC-Biotin MW 567.70 O EZ-Link Sulfo-NHS-LC-LC-Biotin Spacer Arm 30.5 Å 50 mg 21338 NH HN O O 669.75 30.5 + – H Na O O N S EZ-Link Sulfo NHS-LC-LC-Biotin, O S N N H 10 x 1 mg A35358 O O O No-Weigh Format O Sulfo-NHS-LC-LC-Biotin MW 669.75 Spacer Arm 30.5 Å O NH HN O EZ-Link NHS-SS-Biotin 504.65 24.3 H 50 mg 21441 O S N N S S O O O NHS-SS-Biotin MW 504.65 59 Spacer Arm 24.3 Å Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

EZ-Link Sulfo-NHS-SS-Biotin O 100 mg 21331 100 mg PG82077 Pierce Premium Grade NH HN Sulfo-NHS-SS-Biotin 606.69 24.3 O 1 g PG82078 + – H Na O O S N EZ-Link Sulfo-NHS-SS-Biotin, O S N S S 10 x 1 mg A39258 No-Weigh Format O O O O Sulfo-NHS-SS-Biotin O MW F606.69 Spacer Arm 24.3 Å N F F NH EZ-Link NHS-Iminobiotin 436.41 13.5 HN 100 mg 21117 O O N S O O

NHS-Iminobiotin TrifluoroacetamidO e MW 436.41 Spacer Arm 13.5 Å NH HN F EZ-Link PFP-Biotin 410.36 9.6 F O 50 mg 21218 S O F F F

Biotin-PFP Ester O 25 mg 21330 MW 410.36 EZ-Link NHS-PEG -Biotin Spacer Arm 9.6 Å NH 50 mg 21362 4 HN O 1 g 21363 588.67 29.0 H O O O N S EZ-Link NHS-PEG -Biotin, N O O 4 O O 10 x 2 mg A39259 No-Weigh Format O NHS-PEG4-Biotin MW 588.67 O 25 mg 21312 Spacer Arm 29.0 Å EZ-Link NHS-PEG12-Biotin NH 500 mg 21313 O HN 941.09 56 O H EZ-Link NHS-PEG12-Biotin, N N O O S 10 x 1 mg A35389 No-Weigh Format ][ 12 O O

NHS-PEG12-Biotin MW 941.09 O Spacer Arm 56 Å NH HN O EZ-Link NHS-SS-PEG -Biotin 751.94 37.9 H H 50 mg 21442 4 O S N O O N S N S O O O O O O

Sulfhydryl-reactive

O

NH HN O H EZ-Link BMCC-Biotin 533.68 32.6 O N 50 mg 21900 N S H N O

O Biotin-BMCC MW 533.68 Spacer Arm 32.6 Å O

EZ-Link HPDP-Biotin NH 50 mg 21341 HN O 539.78 29.2 H N S N EZ-Link HPDP-Biotin, S N S H 10 x 1 mg A35390 No-Weigh Format O Biotin-HPDP MW 539.78 Spacer Arm 29.2 Å

60 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

O

NH HN EZ-Link Iodoacetyl-LC-Biotin 510.43 27.1 O 50 mg 21333 H I N N S H O

Iodoacetyl-LC-Biotin O MW 510.43 Spacer Arm 27.1 Å NH HN EZ-Link Iodoacetyl-PEG -Biotin 542.43 24.7 O 50 mg 21334 2 H I O N N O S H O

Iodoacetyl-PEG2-Biotin O MW 542.43 F NH Spacer Arm 24.7 Å HN F O EZ-Link TFP-PEG3-Biotin 694.74 32.6 H H 50 mg 21219 N O O N F O O S F O O

TFP-PEG3-Biotin MW 694.74 Spacer Arm 32.6 Å O EZ-Link Maleimide-PEG -Biotin 50 mg 21901BID 2 NH HN O O 525.62 29.1 H O N EZ-Link Maleimide-PEG -Biotin, N N O S 2 H 10 x 2 mg A39261 No-Weigh Format O O Maleimide-PEG2-Biotin MW 525.62 O Spacer Arm 29.1 Å NH O HN EZ-Link Maleimide-PEG -Biotin 922.09 59.1 25 mg 21911 11 H H N N N O S ][ 11 O O O Maleimide-PEG -Biotin Carboxyl-reactive 11 MW 922.09 Spacer Arm 59.1 Å O

NH HN EZ-Link Biocytin 372.48 20.1 O 100 mg 28022 H N HO S

NH2 O

Biocytin O MW 372.48 Spacer Arm 20.1 Å NH HN EZ-Link Pentylamine-Biotin 328.47 18.9 50 mg 21345 H H N N 2 S O Pentylamine-Biotin O MW 328.47 Spacer Arm 18.9 Å NH HN EZ-Link Amine-PEG2-Biotin 374.50 20.4 50 mg 21346 H H N O N 2 O S O

Amine-PEG2-Biotin O MW 374.50 NH Spacer Arm 20.4 Å HN EZ-Link Amine-PEG -Biotin 418.55 22.9 50 mg 21347 3 H H N O N 2 O O S O

Amine-PEG3-Biotin MW 418.55 Spacer Arm 22.9 Å

61 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No.

H N O EZ-Link Amine-PEG4-Desthiobiotin, O 432.55 28.8 H N O O 5 x 1 mg A39263 No-Weigh Format 2 O O N N H H

Amine-PEG4-Desthiobiotin O

NH MW 432.55 HN Spacer Arm 28.8 Å EZ-Link Amine-PEG -Biotin 770.97 53.2 H N H 100 mg 26136 11 2 O O O O O N O O O O O O S O

Amine-PEG11-Biotin Carbonyl-reactive MW 770.97 Spacer Arm 53.2 Å O

NH HN EZ-Link Hydrazide-Biocytin 386.51 19.7 O 25 mg 28020 H H N N 2 N S H NH2 O

Biocytin-HO ydrazide MW 386.51 NH SpacerHN Arm 19.7 Å EZ-Link Hydrazide-Biotin 258.34 15.7 100 mg 21339 H N S H2N O

Biotin-Hydrazide O MW 258.34 NH Spacer Arm 15.7 Å HN EZ-Link Biotin-LC-Hydrazide 371.50 24.7 O 50 mg 21340 H H N N 2 N S H O

Biotin-LC-Hydrazide O MW 371.50 NH Spacer Arm 24.7 Å HN EZ-Link Hydrazide-PEG -Biotin 505.63 31.3 50 mg 21360 4 H H N O O N S H2N O O O O

Biotin-PEG4-Hydrazide H MW 505.63 N O Spacer Arm 31.3 Å O EZ-Link Hydrazide-PEG4-Desthiobiotin, O 475.58 31.2 H N O O 5 x 1 mg A39262 No-Weigh Format 2 N O O N N H H H

Hydrazide-PEG4-Desthiobiotin MW 475.58O Spacer Arm 31.2 NHÅ HN EZ-Link Alkoxyamine-PEG -Biotin 434.22 27.0 50 mg 26137 4 H O O N S H2N O O O Alkoxyamine-PEG -Biotin 4 O MW 434.22 NH Spacer Arm 27.0 Å HN

EZ-Link Alkoxyamine-PEG12-Biotin 786.97 55.4 H 50 mg 26139 O O O O O O N S H2N O O O O O O O

Alkoxyamine-PEG12-Biotin MW 786.97 Spacer Arm 55.4 Å

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62 Ordering Information (continued) Spacer Product MW arm (Å) Structure Quantity Cat. No. Photoreactive or chemoselective

O O NH HN O EZ-Link Psoralen-PEG3-Biotin 688.79 36.9 H H 5 mg 29986 N O N O O O S O O O

Psoralen-PEG3-Biotin MW 688.79 O Spacer Arm 36.9 Å F NH HN + N F EZ-Link TFPA-PEG -Biotin 663.69 33.4 N 25 mg 21303 3 –N H H N O O N F O S F O O

TFPA-PEG3-Biotin O MW 663.69 H Spacer Arm 33.4 Å N EZ-Link Phosphine-PEG - H3CO O 4 778.87 30.6 H O 5 x 1 mg A39264 Desthiobiotin, No-Weigh Format N O O N Ph2P O O N H H O Phosphine-PEG4-Desthiobiotin MW 778.87 Spacer Arm 30.6 Å

63 Modification reagents for reduction and denaturation of proteins Ordering Information (continued) Spacer Product MW arm (Å) Reactive group Structure Quantity Cat. No. Disulfide bond reduction Pierce 2-Mercaptoethanol/ 78.13 NA Thiol OH 10 x 1 mL 35602 (β-mercaptoethanol) HS Pierce Mercaptoethylamine∙HCl +NH Cl– 78.13 NA Thiol HS 3 6 x 6 mg 20408 (2-mercaptoethylamine∙HCl (2-MEA)) 2-Mercaptoethylamine•HCI M.W.O 113.61 HO Pierce Cysteine·HCl 175.63 NA Thiol + — 5 g 44889 NH3 Cl H2O HS

Cysteine•HCl•H20 Pierce DTT, Cleland’s Reagent/ M.W. 175.6 5 g 20290 Dithiothreitol OH HS 154.25 NA Thiol SH

Pierce DTT, No-Weigh Format OH 48 x 7.7 mg A39255 DTT M.W. 154.25 OH

O O

Bond-Breaker TCEP Solution, 286.25 NA Phosphine HO P 5 mL 77720 Neutral pH

O OH TCEP M.W. 250.15 1 g 20491 Pierce TCEP∙HCl O OH O OH 10 g 20490 SH 286.65 NA Phosphine 1 g RS PG82080O R + R S + HH O HO P OH HO P OH + R Pierce Premium Grade TCEP∙HCl 10 g PG82089 HS O O O O TCEP 100 g PG82090 Schiff base reduction to alkylamine linkage

AminoLink Reductant + 62.84 NA Cyanoborohydride N BH 3–Na 2 x 1 g 44892 (sodium cyanoborohydride) Sodium cyanoborohydride Protein denaturants and chaotropes M.W. 62.84 Guanidine·HCl 500 g 24110 H2N C NH 2·HCl 95.53 NA NA Guanidine·HCl (8 M solution) NH 200 mL 24118 Guanidine Hydrochloride O Lorem ipsum M.W. 95.54 Urea 60.06 NA NA 1 kg 29700 H2N NH2 Urea M.W. 60.06

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64 Modification reagents for proteins and peptides

Ordering Information (continued) Spacer Product MW arm (Å) Reactive group Structure Quantity Cat. No. Irreversibly blocks primary amines

O + – Pierce Sulfo-NHS-Acetate Na O O S (sulfo-N-hydroxysulfosuccinimide 259.17 NA NHS ester O N 100 mg 26777 acetate) O O O

Reversibly blocks primary amines Sulfo-NHS acetate MWO 259.17 H3C Pierce Citraconic Anhydride 112.08 NA NA O 100 g 20907 (2-methylmaleic anhydride)

O

Protects and extends primary amines Citraconic Anhydride M.W. 112.08

O Pierce TFCS NHS ester/ O F H F (N-(ε-Trifluoroacetylcaproyloxy) 324.25 7.7 trifluoroacetyl- N N 50 mg 22299 O F succinimide ester) protected –NH2 O O

Modifies primary amines to contain a protected sulfhydryl group TFCS 6-(N-Trifluoroacetyl)caproic acid succinimide O MW 324.25 Pierce SATP OO Spacer Arm 7.7 Å (N-succinimidyl S-acetylthio- 245.25 4.1 NHS ester N 50 mg 26100 propionate) OS O SATP

NHS ester/ O M.W. 245.25 Pierce SAT(PEG) 4 acetylated SpacerO Arm 4.1 Å O (N-succinimidyl S-acetyl(thio- 421.46 18.3 N O O 100 mg 26099 sulfhydryl OO O S tetraethylene glycol) O (protected)

O O Pierce SATA 231.23 2.8 NHS ester N S 50 mg 26102 (N-succinimidyl S-acetylthioacetate) O O O

Modifies primary amines to contain a free sulfhydryl group SATA MW 231.23 SpacerS arm+ 2.8– Å Traut’s Reagent NH2 Cl 137.6 3 8.1 Iminothiolane 500 mg 26101 (2-iminothiolane) Traut’s reagent Adds amine or carboxylic acid functional group to protein or surface MW 137.63 Spacer arm 8.1 Å Pierce AEDP O Amine/ (3-((2-aminoethyl)dithio)propionic 217.74 NA S 50 mg 22101 carboxylic acid - + acid∙HCl) Cl H3 N S OH

Irreversibly blocks sulfhydryl groups AEDP M.W. 217.74 O Spacer Arm 9.5 Å CH Pierce NEM 3 125.13 NA Maleimide N 25 g 23030 (N-ethylmaleimide)

O NEM N-Ethylmaleimide MW 125.13

65 Ordering Information (continued) Spacer Product MW arm (Å) Reactive group Structure Quantity Cat. No. Reversibly blocks sulfhydryl groups

OS Pierce MMTS CH3 126.20 NA NA S 200 mg 23011 (methyl methanethiosulfonate) H3C O

Adds primary amine to glass and silica surfaces through silylation MMTS MW 126.20

Pierce APTS 221.37 NA NA O O 100 g 80370 (3-aminopropyltriethoxysilane) Si NH O 2

Oxidizes carbohydrates for reductive amination APTS M.W. 221.37 Na+ O– Pierce Sodium Meta-Periodate 213.89 NA Periodate O I O 25 g 20504 O Sodium meta-Periodate Alkylates reduced cysteines M.W. 213.89 O Pierce Iodoacetic Acid 185.95 NA Iodoacetyl I 500 mg 35603 OH Iodoacetic Acid O Pierce Iodoacetamide, M.W. 185.95 184.96 NA Iodoacetyl 30 x 9.3 mg A39271 No-Weigh Format I NH2 Iodoacetamide M.W. 184.96O Pierce Chloroacetamide, 93.51 NA Chloroacetyl 10 x 1 mg A39270 No-Weigh Format CI NH2 Deprotects SATA-modified molecules

– + Pierce Hydroxylamine·HCl 69.49 NA NA Cl H3N OH 25 g 26103 Hydroxylamine Hydrochloride MW 69.49

66 PEGylation (PEG labeling) reagents for proteins O O

Ordering Information (continued) N CH3 MS(PEG)n O O Spacer Methyl-PEGn-NHS ester O n Succinimidyl-([N-methyl]-#ethyleneglycol) ester Product MW arm (Å) Reactive group Structure Quantityn = 8 Cat. No. Amine-reactive linear PEGylation of protein or surface, terminating with a methyl group MS(PEG)8 16.4 Å MW 509.4 Spacer arm 30.8 Å O 100 mg 22341 MS(PEG) O 4 333.33 16.4 NHS ester N O O n = 12 O O O CH (methyl-PEG4-NHS ester) 3 MS(PEG) 1 g 1222342 O MW 685.71 Spacer arm 44.9 Å O MS(PEG)4 O MW 333.33 MS(PEG)8 n = 24 509.40 30.8 NHS ester N SCHpacer3 arm 16.4 Å 100 mg 22509 (methyl-PEG -NHS ester) O O MS(PEG) 8 8 24 O MW 1,214.39 Spacer arm 88.2 Å O O 100 mg 22685 MS(PEG)12 685.71 44.9 NHS ester N CH3 O O (methyl-PEG12-NHS ester) O 12 1 g 22686

O O MS(PEG)24 1,214.39 88.2 NHS ester N CH3 100 mg 22687 O O (methyl-PEG24-NHS ester) Lorem ipsum O 24

Amine-reactive branched PEGylation of a protein or surface, terminating with a methyl group

O

N O O O

O NH

O 46.2Å TMS(PEG) O 12 25.5Å H O O N O O O O O O 2,420.80 77.5 NHS ester O O O O O O CH3 1 g 22424 ((methyl-PEG ) -PEG -NHS ester) O 12 3 4 O O

O O O O O O CH3 O N O O O O O O O N H H O H N O O O O O O O O O O O O CH3 O

52.0Å

Sulfhydryl-reactive branched PEGylation of a protein or surface, terminating with a methyl group

O H MM(PEG)12 710.81 51.9 Maleimide N N CH3 100 mg 22711 O (methyl-PEG12-maleimide) O O 12 O NH2 O HO O CA(PEG) n H n MM(PEG)24 CA(PEG)n 1,239.44 95.3 Maleimide CA(PEGN )n CaNrboxy-PEGCHn-A3 mine 100 mg 22713 n O (methyl-PEG24-maleimide) Carboxy-PEGCan-Arbmineoxyl-(#eth yleneglycol) ethylamine n = 8 Carboxy-PEGn-Amine Carboxyl-(#ethyleneglyO col) ethyO lamine 24 CA(PEG)8 Carboxyl-(#ethyleneglycol) ethylamine MW 441.51 O PEGylation of a protein or surface, terminating with a carboxylicO acid or primary amine 18.1Å Spacer Arm 33.6Å O O O O HO O O NH2 HO O O O NH2 n = 12 CA(PEG)4 100 mg 26120 Amine/ CA(PEG)4 O O CA(PEG) (carboxyl-(4-ethyleneglycol) 265.30 18.1 CA(PEG)4 12 M.W.HO 265.30 O O NH2 carboxylic acid M.W. 265.30 MW 617.72 ethylamine) Spacer Arm 18.1 Å 1 g 26121 Spacer Arm 18.1 Å Spacer Arm 46.8Å

CA(PEG)4

HO

HO CA(PEG) CA(PEG) HO MW 265.30 100 mgn = 24 26122 8 CA(PEG)8 O Amine/CA (PEG)8 O NH

(carboxyl-(8-ethyleneglycol) 441.51 33.6 M.W. 441.51 ][ SpacerNH Arm2 18.1Å CA(PEG)24

M.W. 441.51 O ][ 8 NH2

O ][ carboxylicSpacer Ar macid 33.6 Å O 8 ethylamine) Spacer Arm 33.6 Å 1 g MW 1146.3526123

Spacer Arm 89.8Å

HO

HO CA(PEG) HO CA(PEG) CA(PEG)12 O 100 mg 26124 12 CA(PEG)12 O NH

Amine/M. W. 617.72 ][ NH2

M.W. 617.72 O ][ 12 NH2 (carboxyl-(12-ethyleneglycol) 617.72 46.8 O ][ O 12 carboxylicSpacer Ar macid 46.8 Å 12

ethylamine) Spacer Arm 46.8 Å 1 g 26125

HO

HO CA(PEG) HO CA(PEG)24 O CA(PEG) CA(PEG)24 O NH 100 mg 26126

24 M.W. 1146.35 ][ NH2

Amine/M. W. 1146.35 O ][ 24 NH2

O ][ (carboxyl-(24-ethyleneglycol) 1,146.35 89.8 Spacer Arm 89.8 Å O 24 carboxylicSpacer Ar macid 89.8 Å ethylamine) 1 g 26127

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67 Ordering Information (continued) Spacer Product MW arm (Å) Reactive group Structure Quantity Cat. No. PEGylation of a gold, silver, or metal surface, terminating with a carboxylic acid or methyl group 55.5 Å O S CL(PEG) S 12 Carboxylic acid/ H Carboxy-PEG-Lipoamide 806.03 55.5 N 100 mg 26135 bidentate thiol HO O Compound 12 O

CL(PEG)12 O CT(PEG)12 Carboxylic acid/ 634.77 47.8 O CarbOoxy-PEGO -LipoamideO O O 100 mg 26133 Carboxy-PEG-Thiol Compound thiol HO O O O 12 O O O SH MW CT80(PEG6.)1203 Ca23.6rboxy-PE ÅG12-Thiol Spacer ArMW m634.77 55.5 Å S S Spacer Arm 47.8 Å H ML(PEG)4 O O N 395.58 23.6 Bidentate thiol H C O O H2N 100CH3 mg 26134 Methyl-PEG-Thiol Compound 3 O MA(PEG)n O n Methyl-PEGML(Pn-AminEG)e4 PEGylation of a protein or inert material surface, terminating with a methyl group Methyl-PEG4-Lipoamide n = 8 Methyl-(#ethylenegly15.8 Åcol) amine MW 395.58 MA(PEG)8 Spacer Arm 23.6 Å MT(PEG) O O SH MW 383.48 4 224.32 15.8 Thiol H C O O 100 mg 26132 Methyl-PEG-Thiol Compound 3 15.5 Å Spacer Arm 29.7 Å PEGylation of a protein, oxidized carbohydrate, or surface, terminating with a methyl groupMT(PEG)4 n = 12 H N O O 2 Methyl-PEG4-Thiol MA(PEG)12 O O CH3 MA(PEG) MW 224.32 MW 559.69100 mg 26110 4 Spacer Arm 43.9 Å (methyl-(4-ethyleneglycol) 207.27 15.5 Amine SMApacer(PEG Ar)4m 15.8 Å ethylamine) MW 207. 27 n = 241 gLorem ipsum26111 Spacer Arm 15.5 Å MA(PEG)24 MW 1088.32 MA(PEG)8 100 mg 26112 H2N CH3 Spacer Arm 86.1 Å (methyl-(8-ethyleneglycol) 383.48 29.7 Amine O ethylamine) 8 1 g 26113

MA(PEG)12 100 mg 26114 H2N CH3 (methyl-(12-ethyleneglycol) 559.69 43.9 Amine O ethylamine) 12 1 g 26115

MA(PEG)24 100 mg 26116 H2N CH3 (methyl-(24-ethyleneglycol) 1,088.32 86.1 Amine O ethylamine) 24 1 g 26117

68 Fluorescent labeling reagents and kits

Ordering Information (continued) Emission Product Ex/Em color Quantity Cat. No. Amine-reactive Alexa Fluor 350 NHS Ester (Succinimidyl Ester) 346/442 Blue 5 mg A10168 Alexa Fluor 350 Antibody Labeling Kit 346/442 Blue 1 kit A20180 1 mg A30000 Alexa Fluor 405 NHS Ester (Succinimidyl Ester) 402/421 Blue 5 mg A30100 Pacific Blue Succinimidyl Ester 410/455 Blue 5 mg P10163 7-Diethylaminocoumarin-3-Carboxylic Acid, Succinimidyl Ester 375/470 Blue 25 mg D1412 BODIPY 493/503 NHS Ester (Succinimidyl Ester) 500/506 Green 5 mg D2191 BODIPY FL NHS Ester (Succinimidyl Ester) 505/513 Green 5 mg D2184 Fluorescein-5-Isothiocyanate (FITC “Isomer I”) 494/518 Green 1 g F143 3 x 100 µg A37570 Alexa Fluor 488 TFP Ester 495/519 Green 25 mg A37563 1 mg A20000 Alexa Fluor 488 NHS Ester (Succinimidyl Ester) 495/519 Green 5 mg A20100 Alexa Fluor 488 Protein Labeling Kit 495/519 Green 1 kit A10235 Alexa Fluor 488 Microscale Protein Labeling Kit 495/519 Green 1 kit A30006 Alexa Fluor 488 Antibody Labeling Kit 495/519 Green 5 rxn kit A20181 APEX Alexa Fluor 488 Antibody Labeling Kit 495/519 Green 1 kit A10468 Zip Alexa Fluor 488 Rapid Antibody Labeling Kit 495/519 Green 1 kit Z11233 3 x 100 µg P36012 pHrodo iFL Green STP Ester (amine-reactive) 505/520 Green 1 mg P36013 pHrodo iFL Green Microscale Protein Labeling Kit 505/520 Green 1 kit P36015 Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer 495/524 Green 5 mg O6149 Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 5-isomer 495/524 Green 5 mg O6147 Oregon Green 514 Carboxylic Acid, Succinimidyl Ester 511/530 Yellow 5 mg O6139 Alexa Fluor 514 NHS Ester (Succinimidyl Ester) 517/542 Yellow 1 mg A30002 Alexa Fluor 430 NHS Ester (Succinimidyl Ester) 434/539 Yellow 5 mg A10169 BODIPY R6G NHS Ester (Succinimidyl Ester) 528/550 Orange 5 mg D6180 Pacific Orange Succinimidyl Ester, Triethylammonium Salt 400/551 Orange 1 mg P30253 1 mg A20001 Alexa Fluor 532 NHS Ester (Succinimidyl Ester) 531/554 Orange 5 mg A20001MP BODIPY 530/550 NHS Ester (Succinimidyl Ester) 534/554 Orange 5 mg D2187 3 x 100 µg A37571 1 mg A20009 Alexa Fluor 555 NHS Ester (Succinimidyl Ester) 555/565 Orange 5 mg A20109 25 mg A37564 Alexa Fluor 555 Protein Labeling Kit 555/565 Orange 1 kit A20174 Alexa Fluor 555 Microscale Protein Labeling Kit 555/565 Orange 1 kit A30007 Alexa Fluor 555 Antibody Labeling Kit 555/565 Orange 5 rxn kit A20187 APEX Alexa Fluor 555 Antibody Labeling Kit 555/565 Orange 1 kit A10470 Zip Alexa Fluor 555 Rapid Antibody Labeling Kit 555/565 Orange 1 kit Z11234 BODIPY 558/568 NHS Ester (Succinimidyl Ester) 558/569 Orange 5 mg D2219 BODIPY TMR-X NHS Ester (Succinimidyl Ester) 542/574 Orange/red 5 mg D6117 1 mg A20002 Alexa Fluor 546 NHS Ester (Succinimidyl Ester) 556/575 Orange/red 5 mg A20102 Alexa Fluor 546 Protein Labeling Kit 556/575 Orange/red 1 kit A10237 Alexa Fluor 546 Antibody Labeling Kit 556/575 Orange/red 1 kit A20183 BODIPY 576/589 NHS Ester (Succinimidyl Ester) 576/590 Red 5 mg D2225 3 x 100 μg P36011 pHrodo iFL Red STP Ester (amine-reactive) 566/590 Red 1 mg P36010 Alexa Fluor 568 NHS Ester (Succinimidyl Ester) 578/603 Red 1 mg A20003 5 mg A20103 Alexa Fluor 568 Antibody Labeling Kit 578/603 Red 5 rxn kit A20184 APEX Alexa Fluor 568 Antibody Labeling Kit 578/603 Red 1 kit A10494 Alexa Fluor 568 Protein Labeling Kit 578/603 Red 1 kit A10238

69 Ordering Information (continued) Emission Product Ex/Em color Quantity Cat. No. Amine-reactive BODIPY TR-X NHS Ester (Succinimidyl Ester) 589/617 Red 5 mg D6116 3 x 100 μg A37572 Alexa Fluor 594 NHS Ester (Succinimidyl Ester) 590/617 Red 1 mg A20004 5 mg A20104 Alexa Fluor 594 NHS Ester (Succinimidyl Ester) 590/617 Red 25 mg A37565 Alexa Fluor 594 Protein Labeling Kit 590/617 Red 1 kit A10239 Alexa Fluor 594 Microscale Protein Labeling Ki 590/617 Red 1 kit A30008 Alexa Fluor 594 Antibody Labeling Kit 590/617 Red 1 kit A20185 APEX Alexa Fluor 594 Antibody Labeling Kit 590/617 Red 1 kit A10474 BODIPY 630/650-X NHS Ester (Succinimidyl Ester) 625/640 Far red 5 mg D10000 1 mg A20005 Alexa Fluor 633 NHS Ester (Succinimidyl Ester) 632/647 Far red 5 mg A20105 Alexa Fluor633 Protein Labeling Kit 632/647 Far red 5 rxn kit A20170 BODIPY 650/665-X NHS Ester (Succinimidyl Ester) 646/660 Far red 5 mg D10001 3 x 100 μg A37573 1 mg A20006 Alexa Fluor 647 NHS Ester (Succinimidyl Ester) 650/668 Far red 5 mg A20106 25 mg A37566 Alexa Fluor 647 Protein Labeling Kit 650/668 Far red 1 kit A20173 Alexa Fluor 647 Microscale Protein Labeling Kit 650/668 Far red 1 kit A30009 Alexa Fluor 647 Antibody Labeling Kit 650/668 Far red 5 rxn kit A20186 APEX Alexa Fluor 647 Antibody Labeling Kit 650/668 Far red 1 kit A10475 Zip Alexa Fluor 647 Rapid Antibody Labeling Kit 650/668 Far red 1 kit Z11235 3 x 100 μg A37574 1 mg A20008 Alexa Fluor 680 NHS Ester (Succinimidyl Ester) 679/702 Far red 5 mg A20108 25 mg A37567 Alexa Fluor 680 Protein Labeling Kit 679/702 Far red 1 kit A20172 Alexa Fluor 680 Antibody Labeling Kit 679/702 Far red 5 rxn kit A20188 1 mg A20010 Alexa Fluor 700 NHS Ester (Succinimidyl Ester) 702/723 NIR 5 mg A 20110 Alexa Fluor 790 NHS Ester (Succinimidyl Ester) 702/723 NIR 100 μg A30051 Alexa Fluor 790 Antibody Labeling Kit 702/723 NIR 5 rxn kit A20189 Sulfhydryl-reactive

Alexa Fluor 350 C5 Maleimide 346/442 Blue 1 mg A30505 BODIPY FL Maleimide (BODIPY FL N-(2-Aminoethyl)Maleimide) 410/455 Blue 1 mg B10250 Fluorescein-5-Maleimide 505/513 Green 25 mg F150

Alexa Fluor 488 C5 Maleimide 494/518 Green 1 mg A10254 Oregon Green 488 Maleimide 495/519 Green 1 mg O6034 Alexa Fluor 532 C Maleimide 495/524 1 mg A10255 5 Green Alexa Fluor 555 C2 Maleimide 531/554 1 mg A20346

BODIPY TMR C5 Maleimide 555/565 Orange 1 mg B30466

Alexa Fluor 546 C5 Maleimide 542/574 Orange/red 1 mg A10258 Tetramethylrhodamine-5-Maleimide, single isomer 556/575 Orange/red 1 mg T6027

Rhodamine Red C2 Maleimide 555/580 Orange/red 1 mg R6029

Alexa Fluor 568 C5 Maleimide 570/590 Red 1 mg A20341

Texas Red C2 Maleimide 580/605 Red 1 mg T6008

Alexa Fluor 594 C5 Maleimide 595/615 Red 1 mg A10256

Alexa Fluor 633 C5 Maleimide 590/617 Red 1 mg A20342

Alexa Fluor 647 C2 Maleimide 632/647 Far red 1 mg A20347

Alexa Fluor 680 C2 Maleimide 650/668 Far red 1 mg A20344

Alexa Fluor 750 C5 Maleimide 679/702 NIR 1 mg A30459

70 Ordering Information (continued) Emission Emission/ Product color excitation Quantity Cat. No. Carboxyl-reactive Alexa Fluor 350 Hydrazide 346/442 Blue 5 mg A10439 Alexa Fluor 350 Hydroxylamine 346/442 Blue 1 mg A30627 5-FAM (5-Carboxyfluorescein), single isomer 494/518 Green 100 mg C1359 Fluorescein-5-Thiosemicarbazide 494/518 Green 100 mg F121 Fluorescein Cadaverine 494/518 Green 25 mg A10466 Alexa Fluor 488 Hydrazide 495/519 Green 1 mg A10436 Alexa Fluor 488 Hydroxylamine 495/519 Green 1 mg A30629 Alexa Fluor 488 Cadaverine 495/519 Green 1 mg A30676 Qdot 525 ITK Carboxyl Quantum Dots 405/525 Green 250 µL Q21341MP Qdot 545 ITK Carboxyl Quantum Dots 405/545 Yellow 250 µL Q21391MP Qdot 565 ITK Carboxyl Quantum Dots 405/565 Orange 250 µL Q21331MP Alexa Fluor 555 Hydrazide 555/565 Orange 1 mg A20501MP Alexa Fluor 555 Cadaverine 555/565 Orange/red 1 mg A30677 5-TAMRA (5-Carboxytetramethylrhodamine), single isomer 555/580 Orange/red 10 mg C6121 Qdot 585 ITK Carboxyl Quantum Dots 405/585 Red 250 µL Q21311MP Qdot 605 ITK Carboxyl Quantum Dots 405/605 Red 250 µL Q21301MP 5-ROX (5-Carboxy-X-Rhodamine, Triethylammonium Salt), single isomer 580/605 Red 10 mg C6124 Texas Red Hydrazide, >90% single isomer 595/615 Red 5 mg T6256

Texas Red Cadaverine (Texas Red C5) 595/615 Red 5 mg T2425 Qdot 625 ITK Carboxyl Quantum Dots 405/625 Red 250 µL A10200 Qdot 655 ITK Carboxyl Quantum Dots 405/655 Far red 250 µL Q21321MP Qdot 705 ITK Carboxyl Quantum Dots 405/705 NIR 250 µL Q21361MP Qdot 800 ITK Carboxyl Quantum Dots 405/800 NIR 250 µL Q21371MP Chemoselective SiteClick Antibody Azido Modification Kit NA N/A 1 kit S20026 Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling 494/518 Green 1 each C20027 Alexa Fluor 488 Azide 494/518 Green 0.5 mg A10266 Alexa Fluor 488 Alkyne 494/518 Green 0.5 mg A10267 Click-iT Alexa Fluor 488 sDIBO Alkyne 494/518 Green 0.5 mg C20020 Click-iT Plus Alexa Fluor 488 Picolyl Azide Toolkit 494/518 Green 1 kit C10641 SiteClick Qdot 525 Antibody Labeling Kit 405/525 Green 1 kit S10449 SiteClick Qdot 565 Antibody Labeling Kit 405/565 Orange 1 kit S10450 Alexa Fluor 555 Azide 555/565 Orange/red 0.5 mg A20012 Alexa Fluor 555 Alkyne 555/565 Orange/red 0.5 mg A20013 Click-iT Alexa Fluor 555 sDIBO Alkyne 555/565 Orange/red 0.5 mg C20021 Click-iT Alexa Fluor 555 sDIBO Alkyne for Antibody Labeling 555/565 Orange/red 1 each C20028 Click-iT Plus Alexa Fluor 555 Picolyl Azide Toolkit 555/565 Orange/red 1 kit C10642 SiteClick Qdot 585 Antibody Labeling Kit 405/585 Orange/red 1 kit S10450 SiteClick R-PE Antibody Labeling Kit 565/578 Orange/red 1 kit S10467 Tetramethylrhodamine (TAMRA) Azide 555/580 Red 0.5 mg T10182 Tetramethylrhodamine (TAMRA) Alkyne 555/580 Red 0.5 mg T10183 Click-iT pHrodo iFL Red sDIBO Alkyne for Antibody Labeling 566/590 Red 1 kit C20034 SiteClick Qdot 605 Antibody Labeling Kit 405/605 Red 1 kit S10469 Alexa Fluor 594 Azide 590/617 Red 0.5 mg A10270 Alexa Fluor 594 Alkyne 405/625 Red 0.5 mg A10275 SiteClick Qdot 655 Antibody Labeling Kit 405/655 Far red 1 kit S10453 Alexa Fluor 647 Azide 650/668 Far red 0.5 mg A10277 Alexa Fluor 647 Alkyne 650/668 Far red 0.5 mg A10278 Click-iT Alexa Fluor 647 sDIBO Alkyne 650/668 Far red 0.5 mg C20022 Click-iT Alexa Fluor 647 sDIBO Alkyne for Antibody Labeling 650/668 Far red 1 kit C20029 Click-iT Plus Alexa Fluor 647 Picolyl Azide Toolkit 650/668 Far red 1 kit C10643 SiteClick Qdot 705 Antibody Labeling Kit 405/705 NIR 1 kit S10454 SiteClick Qdot 800 Antibody Labeling Kit 405/800 NIR 1 kit S10455 Related handbooks and resources

The Molecular Probes Handbook

The most complete reference available on fluorescent labeling and detection, The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies contains information on over 3,000 reagents and kits representing the full range of our labeling and detection products. The 11th edition features extensive references, reorganized content, and new technical notes and product highlights.

Download the free handbook at thermofisher.com/molecular-probes-the-handbook

Protein clean-up technical handbook

Learn how to effectively remove contaminants, perform buffer exchange, or concentrate protein samples from 2 µL to 250 mL using various Thermo Scientific™ protein biology tools in this 48-page handbook. Dialyze protein samples securely using Slide-A-Lyzer™ cassettes and devices. Rapidly desalt samples with high protein recovery using Zeba™ desalting spin columns and plates. Efficiently extract specific contaminants using resins optimized for

Protein clean-up detergent or endotoxin removal. Concentrate dilute protein samples quickly technical handbook using Pierce™ Protein Concentrators. Dialysis • Desalting • Detergent removal • Concentration • Endotoxin removal Download the free handbook at thermofisher.com/proteincleanuphandbook

Find out more at thermofisher.com/bioconjugation

For Research Use Only. Not for use in diagnostic procedures. © 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Cy is a registered trademark of GE Healthcare. Hoechst is a trademark of Hoechst GmbH. IRDye is a trademark of LI-COR Biosciences. COL06007 0818