Handbook & Selection Guide

Antibody Production Handbook & Selection Guide

c (#JPTDJFODFTttXXX(#JPTDJFODFTDPN t Protein Estimation Assays t Lysis Bu!ers & Systems t Apoptosis Assays t Protein Fractionation Kits t Cytotoxicity Assays t Dialysis (Micro) System t SAM Methyltransferase Assays t Electrophoresis Clean-Up t Protease Assays t Concentration Systems t Phosphatase Assays t Contamination Removal t Peroxide Assay t Protease Inhibitor Cocktails t Proteomic Grade Detergents t Individual Protease Inhibitors t Research Grade Detergents t Protease Assays t Non-Ionic, Ionic & Zwitterionic t Proteases for Mass Spec. t Detergent Estimations t Sequencing Grade Proteases t Detergent Removal Systems

t 1-Hour Western System t Gel Preparation Chemicals t Transfer Bu!ers & Membranes t Protein Marker Ladders t Membrane Stains t Electrophoresis Bu!ers t Blocking Bu!ers t Reducing & Alkylating Reagents t Secondary Antibodies t Protein Gel Stains t Detection Reagents t Reprobing Reagents t Protein Sample Preparation t A"nity Resins t Protein Clean-Up Systems t 6X His Protein Puri#cation Kits t Electrophoresis Reagents t GST Protein Puri#cation Kits t Mass Spec Grade Protease t Antibody Puri#cation t InGel Digestion Kits t Activated Resins t Peptide Generation Reagents t Bu!ers & Reagents t Biotin Labeling t Carrier Proteins t Cell Surface Protein Labeling t Peptide Coupling Systems t Agarose Coupling Kits t Antibody Puri#cation Resins t Fluorescent Dye Labeling Kits t Antibody Fragmentation Kits t Enzyme Labeling Systems t Coated Plates t Blocking Bu!ers t Homobifunctional t Wash Bu!ers t Heterobifunctional t Secondary Antibodies t Optimizer Systems t Detection Reagents t Cross-Linking Systems t Antibody Labeling Systems

t Apoptosis Assays t DNA Isolation t Cytotoxicity Assays t Transformation & Screening t SAM Methyltransferase Assays t Polymerase Chain Reaction t Protease Assays t Agarose Electrophoresis t Phosphatase Assays t RNA Isolation t Peroxide Assay t Yeast Transformation t ELISA Table of Contents Introduction 2 Antibody Fragmentation 13 The Immune Response...... 2 Immobilized Papain...... 13 Immobilized Pepsin ...... 13 Antibody Production 3 Immobilized Ficin ...... 13 Carrier Proteins Fab Fragmentation ...... 13 KLH ...... 3 F(ab)2 Fragmentation ...... 14 ...... 14 Bovine Serum Albumin (BSA) ...... 3 Fab & F(ab)2 Fragmentation of Mouse IgG1 HyperCarrier™ ...... 4 Antibody Labeling 15 Activated Carrier Proteins4 ActiveHOOK™ Carrier Proteins ...... 4 Fluorescent Dye Labeling5 HOOK™ Dye Labeling Kit (5/6) TAMRA-SE (Rhodamine) ...... 15 Peptide Coupling Kits5 HOOK™ Dye Labeling Kit (FITC) ...... 15 HOOK™ Peptide Coupling (Amine Reactive) ...... 5 OneQuant™ Fluorescent Reagents ...... 15 HOOK™ Peptide Coupling (Sulfhydryl Reactive) ...... 5 Biotin Labeling Antibody Puri!cation 6 Biotin Selection Guide ...... 17 OneQuant™ Biotin Reagents ...... 19 Protein A & Protein G HOOK™ Biotin Kits ...... 19 Immobilized Protein A ...... 6 Micro HOOK™ Biotin Kits ...... 20 Immobilized Protein G ...... 6 HOOK™ BiotinQuant ...... 20 Pearl™ Puri!cation HOOK™ IgG Biotinylation ...... 20 Pearl™ IgG Puri!cation Resin ...... 7 HOOK™ BiotinQuant ...... 21 Pearl™ IgG Puri!cation Kits ...... 7 Avidin ...... 21 Pearl™ Monoclonal IgG Puri!cation Kit ...... 7 HABA ...... 21 Pearl™ Antibody Clean Up Kit ...... 8 Streptavidin Resin ...... 21 IgA Puri!cation Avidin Resin ...... 22 Immobilized Jacalin ...... 8 Monomeric Avidin Resin...... 22 Jacalin, Lyophilized ...... 8 &O[ZNF-BCFMJOH Thiophilic Adsorption HOOK™ HRP PLUS Labeling ...... 23 Thiophilic Resin ...... 8 HOOK™ HRP Sulfo Labeling ...... 23 HOOK™ AP Sulfo Labeling ...... 23 "óOJUZ$PMVNO(FOFSBUJPO9 Sulfhydryl Coupling Resin ...... 9 Antibody Puri!cation Accessories 23 Sulfhydryl Immobilization Kit for Proteins ...... 9 Sulfhydryl Immobilization Kit for Peptides ...... 9 %JTQPTBCMF$PMVNOT Amine Coupling Resin ...... 10 Spin Column, <0.1ml...... 23 CDI Amine Reactive Resin ...... 10 Spin Column, 1ml ...... 23 Carboxyl Coupling Resin ...... 11 Spin Column, 2ml ...... 24 SDC™ (Steroid/Drug/Compound) Immobilization ...... 11 Spin Column, 3ml ...... 24 $PBUFE8FMM1MBUFT Spin Column, 5ml ...... 24 Spin Column, 10ml ...... 24 Well-Coated™ Protein A, Protein G & Protein A/G...... 12 Well-Coated™ Protein L ...... 12 Well-Coated™ Antibody ...... 12

Updated: Jul 28, 2011 For further details, visit www.GBiosciences.com 1 Introduction Antibodies, in particular the production of speci!c antibodies, in the acidi!ed endosomes and lysosomes, digest the complex into have been essential in the advancement of many scienti!c !elds, small peptides (4). particularly proteomics, immunology and cell biology. The immature MHC class II (MHCII) proteins wait for the detection A common practice for the generation of speci!c antibodies is the of foreign material in the endoplasmic reticulum, where it is in a use of immunogenic peptides derived from the protein of interest. complex with the Invariant chain (Ii) (5). Upon detection of the foreign These peptides are short sequences of amino acids that are antigenic, material, the MHCII:Ii complex leaves the ER, traverses the Golgi however they lack elements required for T cell activation. Activation and endosomes and !nally fuses with the MHCII compartment (6). is achieved with the use of “carrier proteins” that are coupled to the Cathepsins, in the MHCII compartment, partially digest Ii chain leaving small antigenic peptide. CLIP in the peptide-binding site. The endosome/lysosome, with the carrier protein:peptide The Immune Response peptides, fuses with the MHCII compartment and a human leukocyte The coupling of the peptide to a carrier protein is important in the antigen (HLA) protein replaces CLIP with a carrier protein:peptide stimulation of the immune response and the generation of antibodies. peptide (7). The mature, peptide-loaded MHC class II protein moves Below is a brief description and diagram summarizing the immune to the plasma membrane and is presented on the cell surface (8). pathway responsible for detecting the peptide and carrier protein and A speci!c helper T-cell binds the peptide-loaded MHC class generating subsequent antibodies. II protein through its T-cell receptor and CD4 receptor (9), which The major histocompatibility complex (MHC) II pathway is stimulates the macrophage to release interleukin-1 (IL-1). In turn, the responsible for the detection and generation of antibodies against helper T-cell releases IL-2 that stimulates itself and the macrophage foreign material, such as the carrier protein:peptide complex. (10). This stimulation and release of further interleukins and A B-cell or a circulating macrophage (antigen presenting cell (APC)) lymphokines leads to the proliferation of the macrophages and the travels through the host’s blood stream in search of foreign material helper T-cells, producing clones and some memory helper T-cells (carrier protein:peptide complex)(1). Once detected, the macrophage (important for fast secondary response). The released interleukins captures the carrier protein:peptide complex and brings it inside stimulate B lymphocyte cells (11) to initiate mitosis and cloning the cell, by a process known as endocytosis (2). The endosome, (12), producing more B lymphocyte cells and some memory B-cells containing the carrier protein:peptide complex, moves towards the (important for fast secondary response). After cloning, the B center of the cell and becomes acidi!ed (3), at which time it may fuse lymphocyte cells enlarge and develop an extensive endoplasmic with the lysosomes, which contain acid proteases. The acid proteases, reticulum and then begin mass-producing antibodies (13).

Figure 1: The Immune Response and Antibody production. 2 For further details, visit www.GBiosciences.com Antibody Production CARRIER PROTEINS KLH """"""""""""""""" """"""""""""""""" For the successful generation of antibodies Keyhole Limpet Hemocyanin Many proteins are suitable for the role as a carrier protein and Keyhole limpet hemocyanin is a widely used carrier protein due to it is their properties that determine, to a large extent, the immune its large molecular mass. It is 4.5x106-1.3x107Da aggregates composed response and outcome of antibody production. Several factors are of 350-390kDa subunits. The large size results in a large number important to consider in the choice of the carrier protein. The !rst is of primary amines that can react with cross-linkers for the coupling the size of the carrier protein. Larger proteins (>60kDa) are preferable of peptides. Supplied as KLH or ActiveHOOK™ KLH, a maleimide as it is highly probable that they contain the elements required for activated carrier protein, as 8 x 2mg single use OneQuant™ vials or T-cell activation and they have multiple and su"cient numbers of 10mg vials. exposed residues for peptide coupling, such as amine and sulfhydryl FEATURES groups. An additional important factor in the choice of a carrier t )JHINPMFDVMBSNBTT Y5-1.3x107Da aggregates) protein is to ensure that the carrier protein of choice is non-self, in fact t 4USPOHFSJNNVOFSFTQPOTFDPNQBSFEUP#4" the more genetically distinct the source the greater likelihood of a t $POWFOJFOUYNHTJOHMFVTF0OF2VBOU™ vials or 10mg vials larger immunogenic response. Keyhole Limpet Hemocyanin (KLH) is a commonly used carrier APPLICATIONS protein because it is puri!ed from a mollusk (a gastropod) and is t $BSSJFSQSPUFJOGPSQFQUJEFTGPSUIFQSPEVDUJPOPGBOUJCPEJFT therefore very genetically distinct from the mammals used in antibody $BU Description Size production. It is highly aggregated, giving it a molecular weight of 786-088 KLH (Immunological Grade) 10mg 4.5x105-1.3x107 kDa and has a large number of available lysine groups. 786-091 OneQuant™ KLH (Immunological Grade) 8 x 2mg Common problems with using KLH as a carrier protein are a result ™ of its large degree of aggregation, which can lead to insolubility in 786-089 ActiveHOOK KLH 10mg aqueous solutions, and the large number of coupling sites which can 786-094 OneQuant™ ActiveHOOK™ KLH 8 x 2mg lead to overloading of the antigenic peptide resulting in precipitation. Bovine Serum Albumin (BSA) is another common carrier protein. Bovine Serum Albumin (BSA) It is smaller than KLH (67kDa), but still immunogenic. BSA is rich in """"""""""""""""" lysine residues (59) of which 30-35 are available for coupling, it is BSA is a single polypeptide of 67kDa and consists of 59 lysine highly soluble and stable making its preparation and use very simple. residues, of which 30-35 have primary amines that can react with Over the years, immunological researchers have focused their crosslinkers for the coupling of peptides. Supplied as BSA or attention on trying to understand the antigen recognition pathways ActiveHOOK™ BSA, a maleimide activated carrier protein, as 8 x 2mg and subsequent immunogenic responses, including antibody single use OneQuant™ vials or 10mg vials. production. Researchers were able to demonstrate that a cationized FEATURES form of BSA, produced by replacing anionic side chain carboxylic t MZTJOFSFTJEVFTBSFDBQBCMFPGDPOKVHBUJOH groups with aminoethylamide groups, was more immunogenic than t 4JOHMFQPMZQFQUJEFQSPUFJO L%B normal BSA1. They have shown that the amount of cationized BSA t .PSFTPMVCMF CVUMFTTJNNVOPHFOJD UIBO,-) (cBSA) required for stimulation of T-cell proliferation in-vitro was 500 t $POWFOJFOUYNHTJOHMFVTF0OF2VBOU™ vials or 10mg vials times less than normal BSA (nBSA), whereas in-vivo cBSA produced responses which were at least twice nBSA and lasted for longer APPLICATIONS periods of time. In addition, antibodies were produced in response to t $BSSJFSQSPUFJOGPSQFQUJEFTGPSUIFQSPEVDUJPOPGBOUJCPEJFT cBSA, in the absence of adjuvants, which was not the case for nBSA. $BU Description Size Further research demonstrated that cBSA exhibited unique immunogenic properties as a result of alterations in the self-regulation 786-086 Bovine Serum Albumin (BSA) (Immunological Grade) 10mg ™ of the immune response2. Pretreatment with cBSA, either orally3 or 786-090 OneQuant BSA (Immunological Grade) 8 x 2mg intravenously, prior to immunization with cBSA greatly enhanced the 786-087 ActiveHOOK™ BSA 10mg anti-BSA response; nBSA pretreatment suppressed this response. 786-093 OneQuant™ ActiveHOOK™ BSA 8 x 2mg The underlying mechanisms to the increased strength and duration of antibody responses are not fully understood. Researchers, however, believe that the increased positive charge (pI>10.5) of cBSA gives it greater a"nity for the negative membrane surface of antigen presenting cells (APCs)4 and have shown that cBSA is taken into the cell by an adsorptive mechanism, such as receptor mediated endocytosis, as opposed to the slower #uid phase pinocytosis utilized by nBSA5. This results in a more rapid and e"cient uptake and subsequent processing of the antigen. Interestingly, the enhanced immune response of cBSA can be extended to peptides and proteins coupled to cBSA, allowing for a greater immune response and therefore higher titer antibody production. HyperCarrier™ is a cationized BSA REFERENCES 1. Muckerheide, A., et al (1987) J. Immunol. 138: 833 2. Muckerheide, A., et al (1987) J. Immunol. 138: 2800 3. Domen, P.L., et al (1987) J. Immunol. 139: 3195 4. Dohlman, J.G.,et al (1991) Biochem. Biophys. Res. Commun. 181: 787 5. Apple, R.J.,et al. (1988) J. Immunol. 140: 3290

For further details, visit www.GBiosciences.com 3 Antibody Production HyperCarrier™ ACTIVATED CARRIER PROTEINS """"""""""""""""" """"""""""""""""" A cationized BSA for a greater immune response ™ HyperCarrier™ is normal bovine serum albumin (BSA) that has been ActiveHOOK Carrier Proteins treated with ethylene diamine, which substitutes anionic carboxyl """"""""""""""""" groups with cationic aminoethyl-amide groups. Malemide activated carrier proteins Researchers demonstrated that cationized BSA was more The ActiveHOOK™ line of carrier proteins are maleimide activated immunogenic than normal BSA (1). They have shown that the amount by the addition of sulfoSMCC cross-linker. The ActiveHOOK™ line of cationized BSA (cBSA) required for stimulation of T-cell proliferation rapidly couples to free sulfhydryl groups on peptides and proteins. in-vitro was 500 times less than normal BSA (nBSA), whereas in-vivo These activated carrier proteins save both time and money as separate cBSA produced responses which were at least twice nBSA and lasted cross-linkers are not required and the reaction is a one step reaction. for longer periods of time. In addition, antibodies were produced in See individual carrier protein listings for more information. response to cBSA, in the absence of adjuvants, which was not the case for nBSA. Further research demonstrated that cBSA exhibited unique immunogenic properties as a result of alterations in the self-regulation of the immune response (2). Pretreatment with cBSA, either orally (3) or intravenously, prior to immunization with cBSA greatly enhanced the anti-BSA response; nBSA pretreatment suppressed this immune response. Supplied as HyperCarrier™ or ActiveHOOK™ HyperCarrier™, a maleimide activated carrier protein, as 8 x 2mg single use OneQuant™ vials or 10mg vials. FEATURES t *ODSFBTFECJOEJOHUPJNNVOFTZTUFNDFMMT t 4USPOHFSJNNVOFSFTQPOTFDPNQBSFEUP#4" t 4JOHMFQPMZQFQUJEFQSPUFJO L%B ™ t $POWFOJFOUYNHTJOHMFVTF0OF2VBOU vials or 10mg vials Figure 2: The coupling scheme for ActiveHOOK™ Carrier proteins.

APPLICATIONS $BU Description Size t $BSSJFSQSPUFJOGPSQFQUJEFTGPSUIFQSPEVDUJPOPGBOUJCPEJFT 786-087 ActiveHOOK™ BSA 10mg REFERENCES 786-093 OneQuant™ ActiveHOOK™ BSA 8 x 2mg 1. Muckerheide, A., et al (1987) J. Immunol. 138: 833 786-097 ActiveHOOK™ HyperCarrier™ 10mg 2. Muckerheide, A., et al (1987) J. Immunol. 138: 2800 3. Domen, P.L., et al (1987) J. Immunol. 139: 3195 786-095 OneQuant™ ActiveHOOK™ HyperCarrier™ 8 x 2mg 786-089 ActiveHOOK™ KLH 10mg $BU Description Size 786-094 OneQuant™ ActiveHOOK™ KLH 8 x 2mg 786-096 HyperCarrier™ (Immunological Grade) 10mg 786-092 OneQuant™ HyperCarrier™ (Immunological Grade) 8 x 2mg 786-097 ActiveHOOK™ HyperCarrier™ 10mg 786-095 OneQuant™ ActiveHOOK™ HyperCarrier™ 8 x 2mg

4 For further details, visit www.GBiosciences.com Antibody Production PEPTIDE COUPLING KITS HOOK™ Peptide Coupling """"""""""""""""" (Sulfhydryl Reactive) HOOK™ Peptide Coupling """"""""""""""""" This kit is designed for the coupling of peptides to carrier proteins, (Amine Reactive) utilizing a sulfhydryl group in the peptide. """"""""""""""""" This kit exploits the chemical cross-linker sulfoSMCC to couple Designed for the coupling of peptides to carrier proteins, utilizing peptides through their sulfhydryl groups, found on cysteine side the primary amines and carboxyl groups of the peptides and carrier chains, to the primary amines on the carrier proteins. proteins. This kit utilizes the chemical heterobifunctional crosslinker The N-hydroxysuccinimide (NHS) ester in sulfoSMCC reacts with EDC to couple peptides to carrier proteins. EDC !rst reacts with primary amines to form covalent amide bonds and the maleimide the carboxyl groups, producing an amine reactive intermediate, group reacts with sulfhydryl groups to form stable thioether bonds. O-acylisourea that rapidly reacts with the amine groups of the If peptides do not contain a sulfhydryl group then G-Biosciences peptide/protein. recommends and supplies Traut’s reagent (2-IminothiolaneHCl). ™ This kit utilizes Tube-O-Reactor , which allows for the reactions to Traut’s reagent modi!es primary amines, located at the N-terminus be performed in a single tube, with no loss of essential reagents and and on lysine side chains, introducing a sulfhydryl group that is fully minimum hands on time and e$ort. compatible with this kit. FEATURES This kit utilizes Tube-O-Reactor™, which allows for the reactions to t 4JNQMFUPVTF TJOHMFTUFQSFBDUJPO be performed in a single tube, with no loss of essential reagents and t 4VJUBCMFGPSBMMDBSSJFSQSPUFJOT minimum hands on time and e$ort. APPLICATIONS t $PVQMJOHPGQFQUJEFTUPDBSSJFSQSPUFJOTGPSBOUJCPEZQSPEVDUJPO SULFO3-##

%$# #ARRIER 0ROTEIN #ARRIER 0ROTEIN #OMBINE PEPTIDE CARRIER PROTEIN AND SULFO3-## CROSS 4UBE / $)!,9:%2» TUBE LINKER IN 4UBE / $)!,9:%2» #OMBINE PEPTIDE CARRIER PROTEIN AND %$# CROSSLINKER 4UBE / $)!,9:%2» TUBE IN 4UBE / $)!,9:%2»

!DD $IALYSIS #AP )NCUBATE

4UBE / $)!,9:%2» !DD $IALYSIS #AP )NCUBATE 2EACTION #HAMBER

4UBE / $)!,9:%2» &LOAT )NVERT AND DIALYZE AGAINST 2EACTION #HAMBER SUPPLIED /PTIMIZER "UFFER» TO REMOVE UNCOUPLED PEPTIDE AND CROSS LINKER /PTIMIZER "UFFER» &LOAT )NVERT AND DIALYZE AGAINST 'LASS BEADS TO MIX SUPPLIED /PTIMIZER "UFFER» TO REMOVE UNCOUPLED PEPTIDE Figure 4: HOOK™ Peptide Coupling (Sulfhydryl Reactive) scheme. AND CROSS LINKER /PTIMIZER » "UFFER FEATURES 'LASS BEADS t 4JNQMFUPVTF TO MIX t 4VJUBCMFGPSBMMDBSSJFSQSPUFJOT Figure 3: HOOK™ Peptide Coupling (Amine Reactive) scheme. t 6TFT5VCF03FBDUPS™ for minimum hands on time APPLICATIONS $BU Description Size t $PVQMJOHPGQFQUJEFTUPDBSSJFSQSPUFJOT ™ 786-067 HOOK Peptide Coupling Kit (Amine reactive) 5 Reactions t 4VJUBCMFGPSBOUJCPEZQSPEVDUJPO HOOK™ Peptide Coupling Kit (Amine reactive) 786-068 5 Reactions with Oneant™ BSA $BU Description Size HOOK™ Peptide Coupling Kit (Amine reactive) 786-071 HOOK™ Peptide Coupling Kit (Sulfhydryl reactive) 5 Reactions 786-069 5 Reactions with OneQuant™ KLH HOOK™ Peptide Coupling Kit (Sulfhydryl reactive) 786-072 5 Reactions HOOK™ Peptide Coupling Kit (Amine reactive) with OneQuant™ BSA 786-070 5 Reactions with OneQuant™ HyperCarrier™ HOOK™ Peptide Coupling Kit (Sulfhydryl reactive) 786-073 5 Reactions with OneQuant™ KLH HOOK™ Peptide Coupling Kit (Sulfhydryl reactive) 786-074 5 Reactions with OneQuant™ HyperCarrier™ For further details, visit www.GBiosciences.com 5 Antibody Puri!cation PROTEIN A & PROTEIN G """"""""""""""""" Immobilized Protein A """"""""""""""""" For binding the constant domains of immunoglobulin (Ig) molecules (Table 19). Protein A is coupled to agarose beads by a proprietary coupling method that provides high coupling e"ciency for immunoglobulins and minimal protein A leaching. Immobilized Antibody Protein Protein A Resin is available as resin alone or supplied in a kit format Species Class Protein A Protein G A/G containing: Mouse Total IgG ***** ***** ***** t 5ml resin IgM - - -

t 100ml Protein A or G Binding/Wash Bu$er IgG1 * *** *** (0.1M sodium phosphate, 0.15M NaCl, pH7.5) IgG2a ***** ***** ***** t 100ml Protein A or G Elution Bu$er (100mM glycine, pH3.0) IgG2b ***** ***** ***** t 5 empty 1ml spin columns IgG ***** ***** ***** t 5 empty <5ml gravity #ow columns 3 Human Total IgG ***** ***** ***** The bu$ers are also available separately. IgG ***** ***** ***** FEATURES 1 IgG ***** ***** ***** t 'PSUIFCJOEJOHPGJNNVOPHMPCVMJOT 2 IgG * ***** ***** t )JHICJOEJOHDBQBDJUZNHNMSFTJO 3 IgG ***** ***** ***** t #FBE4USVDUVSFIJHIMZDSPTTMJOLFEBHBSPTF 4 IgM * - * APPLICATIONS IgD - - - t 'PSJNNVOPBóOJUZDISPNBUPHSBQIZJNNVOPQSFDJQJUBUJPO IgA * - * $BU Description Size Fab * * * 786-283 Immobilized Protein A Resin 5ml resin ScFv * - * 786-553 Immobilized Protein A Resin Kit 1 Rat Total IgG * *** ***

786-544 Protein A or G Binding/ Wash Bu$er 100ml IgG1 * *** ***

786-545 Protein A or G Elution bu$er 100ml IgG2a - ***** *****

IgG2b - * * IgG ***** ***** ***** Immobilized Protein G 2c """"""""""""""""" Rabbit Total IgG ***** ***** ***** For binding the constant domains of immunoglobulin (Ig) Goat Total IgG * ***** ***** molecules (Table 19). Protein G is a modi!ed form of Streptococcal IgG1 * ***** ***** group G so that it does not bind to albumin. Protein G is coupled to IgG ***** ***** ***** DSPTTMJOLFEBHBSPTFCFBETCZBQSPQSJFUBSZDPVQMJOHNFUIPE 2 Cat Total IgG ***** * ***** that provides high coupling e"ciency for Ig and minimal protein G Chicken Total IgY - - - leaching. Immobilized Protein G Resin is available as resin alone or supplied in a kit format containing: Cow Total IgG * ***** ***** t 5ml resin IgG1 * ***** *****

t 100ml Protein A or G Binding/Wash Bu$er IgG2 ***** ***** ***** (0.1M sodium phosphate, 0.15M NaCl, pH7.5) Dog Total IgG ***** * ***** t 100ml Protein A or G Elution Bu$er (100mM glycine, pH3.0) Guinea Pig Total IgG ***** * ***** t 5 empty 1ml spin columns Horse Total IgG * ***** ***** t 5 empty <5ml gravity #ow columns IgG(ab) * - * The bu$ers are also available separately. IgG(c) * - * FEATURES IgG(T) - ***** ***** t 'PSUIFCJOEJOHPGJNNVOPHMPCVMJOT Pig Total IgG ***** * ***** t )JHICJOEJOHDBQBDJUZ NH)VNBO*H(NMSFTJO Sheep Total IgG * ***** ***** t -JHBOEEFOTJUZ_NHQSPUFJO(NMSFTJO IgG * ***** ***** t #FBETJ[FɅN 1 IgG ***** ***** ***** t #FBE4USVDUVSFIJHIMZDSPTTMJOLFEBHBSPTF 2 Table 1: Relative a!nity of Protein A, Protein G and Protein A/G for APPLICATIONS immunoglobulins. t 'PSJNNVOPBóOJUZDISPNBUPHSBQIZJNNVOPQSFDJQJUBUJPO

$BU Description Size 786-284 Immobilized Protein G Resin 5ml resin 786-554 Immobilized Protein G Resin Kit 1 786-544 Protein A or G Binding/ Wash Bu$er 100ml 786-545 Protein A or G Elution bu$er 100ml

6 For further details, visit www.GBiosciences.com Antibody Puri!cation PEARL™ PURIFICATION Pearl™ IgG Puri!cation Kits """"""""""""""""" """"""""""""""""" Isolate IgG in physiological bu!er in <15mins ™ Pearl IgG Puri!cation Resin The Pearl™ IgG Puri!cation kits are designed for the one step """"""""""""""""" puri!cation of IgG (Immunoglobulin G) antibodies from serum. The For the one-step puri!ication of the immunoglobulin G (IgG) supplied Pearl™ IgG Puri!cation Resin binds the high abundant, antibodies from serum. The resin binds the high abundant, non-IgG non-IgG proteins (i.e albumin) and allows the IgG molecules to pass proteins (i.e albumin) and allows the IgG molecules to pass through through in a physiological bu$er. The puri!ed IgG molecules can be in a physiological bu$er. The puri!ed IgG molecules can be stored or stored or used in further downstream applications without further used in further downstream applications without further clean-up, clean-up, such as ammonium sulfate precipitation. such as ammonium sulfate precipitation. The Pearl™ IgG puri!cation kits can purify IgG in <15 minutes, which Puri!es IgG in <15 minutes, which is more rapid than the is more rapid than the commonly used Protein A and Protein G resins. commonly used Protein A and Protein G resins. The performance The performance of the Pearl™ IgG Puri!cation Resin is comparable or of the Pearl™ IgG Puri!cation Resin is comparable or better than the better than the Protein A and Protein G resins (Table 18). Protein A and Protein G resins (Table 18). Pearl™ IgG Puri!cation (Spin Format) kit is ideal for the rapid, small scale puri!cation of IgG. The kit is supplied with 3ml Pearl™ IgG Pearl™ IgG Species Puri!cation Resin Protein A Protein G Puri!cation Resin, IgG Isolation Bu$er and 20 spin columns. Suitable for purifying up to 25mg IgG. Mouse ++++ ++++ ++++ Pearl™ IgG Puri!cation kit is supplied with 25ml Pearl™ IgG Human ++++ ++++ ++++ Puri!cation Resin and IgG Isolation Bu$er and is suitable for the Rat ++++ + ++ JTPMBUJPOPG*H(GSPN_NMTFSVN _NH*H( Hamster ++++ ++ ++ Guinea Pig ++++ ++++ ++ $BU Description Size ™ Rabbit ++++ ++++ +++ 786-798 Pearl IgG Puri!cation (Spin Format) kit For 25mg IgG ™ Horse ++++ ++ ++++ 786-799 Pearl IgG Puri!cation kit 'PS_NH*H( Cow ++ ++ ++++ ™ Pig ++++ +++ ++ Pearl Monoclonal IgG Puri!cation Sheep ++ + ++ Kit Goat ++++ + ++ """"""""""""""""" Chicken - - - Isolate monoclonal antibodies from ascites & cell Table 2: Binding e!ciencies of Pearl™ IgG Puri"cation Resin compared to culture supernatant Protein A and Protein G. The Pearl™ Monoclonal IgG Puri!cation kit allows for the rapid puri!cation of antibodies from cell culture supernatant and ascites #uid. The Pearl™ IgG Puri!cation Resin binds the high abundant, non-IgG proteins (i.e. albumin) and allows the IgG molecules to pass through in a physiological bu$er. The IgG molecules can be stored or used in downstream applications without further clean-up, such as Serum Puri!ed IgG withSerum Pearl™ Puri!ed IgG with Pearl™ ammonium sulfate precipitation. IgG (non-reduced) The Pearl™ Monoclonal IgG Puri!cation kit can be used to purify BOUJCPEJFTEJSFDUGSPNDFMMDVMUVSFTVQFSOBUBOUXJUIMFTTUIBO FBS or can be used with ascites #uid after treatment with the supplied IgG (reduced) Ascites PreTreat. The Pearl™.POPDMPOBM*H(1VSJöDBUJPOLJUDBOQVSJGZ*H(GSPN_- IgG (reduced) cell culture supernatant or 200ml ascites #uid. FEATURES t *TPMBUFNPOPDMPOBMBOUJCPEJFTGPSBTDJUFTøVJEPSDFMMDVMUVSF Reduced Non-reduced supernatant Samples Samples t 4VQQMJFEXJUIBTDJUFTQSFUSFBUNFOUSFBHFOUGPSPQUJNBM*H( Figure 5: Pearl™ IgG Puri"cation Resin rapidly puri"es IgG molecules. Rabbit puri!cation serum was dialyzed for 2 hours against IgG Puri"cation Bu#er and treated t 'PS-PGDFMMDVMUVSFTVQFSOBUBOUPS-BTDJUFTøVJE with IgG Puri"cation Resin. The serum and $owthrough were compared under APPLICATIONS reducing and non reducing conditions. t .POPDMPOBMBOUJCPEZJTPMBUJPOGSPNBTDJUFTøVJEPSDFMMDVMUVSF FEATURES supernatant t 4JNQMFTUFQQVSJöDBUJPO $BU Description Size t )JHISFDPWFSZ 1VSJUZ 786-802 Pearl™ Monoclonal IgG Puri!cation Kit 1 APPLICATIONS t 1VSJöDBUJPOPG*H( *NNVOPHMPCVMJO( NPMFDVMFT t 1VSJGZ*H(GSPNTPVSDFTOPUDPNQBUJCMFXJUI1SPUFJO"(

$BU Description Size 786-800 Pearl™ IgG Puri!cation Resin 3ml resin 786-801 Pearl™ IgG Puri!cation Resin 25ml resin

For further details, visit www.GBiosciences.com 7 Antibody Puri!cation Pearl™ Antibody Clean Up Kit THIOPHILIC ADSORPTION """"""""""""""""" """"""""""""""""" Removal of inhibitory BSA & gelatin from antibody solutons Thiophilic Resin """"""""""""""""" Puri!ed and commercial antibodies are routinely stored in bu$ers For thiophilic adsorption of IgG, IgM, IgY and protein containing bovine serum albumin (BSA) and gelatin that act as stabilizers during long term storage. In routine applications, such puri"cation as ELISA, Western blotting and other immunodetection techniques, Thiophilic adsorption or thiophilic chromatography is a these proteins generally do not interfere. The presence of the protein routinely used technique for the low cost, simple puri!cation of stabilizers do interfere with antibody labeling and conjugation immunoglobulins. Thiophilic adsorption was !rst developed by techniques, including biotinylation, #uorescent dye labeling, covalent Porath et al in 1984 and is a group speci!c, salt-dependent puri!cation antibody immobilization and antibody fragmentation experiments. technique that has distinct a"nity towards immunoglobulins and The Antibody Clean Up kit is designed for the rapid clean up of α2-macroglobulins. The thiophilic adsorption works on the principle antibody solutions using a combination of our Pearl™ IgG Puri!cation that some proteins in high salt are able to bind to an immobilized Resin to remove the protein stabilizers and SpinOUT™ desalting ligand that contains a sulfone group in proximity to a thioether group columns to ensure the antibody solutions are in an optimal bu$er for (Figure 160). The bound proteins are then eluted in decreasing salt clean up. The Pearl™ IgG Puri!cation Resin binds the high abundant, concentrations. non-IgG proteins (i.e. BSA and gelatin) and allows the IgG molecules to The thiophilic resin binds immunoglobulins, including IgG, IgY pass through in a physiological bu$er. and IgM, from serum, ascites or tissue culture supernatants and the 'PSUIFQVSJöDBUJPOPGUFONM*H(TBNQMFTXJUIVQUP#4" puri!ed immunoglobulins are then eluted in a near neutral aqueous and gelatin. CVòFS5IFUIJPQIJMJDSFTJOIBTBIJHICJOEJOHDBQBDJUZ _NH FEATURES ml human IgG/ml resin) and a broad speci!city for various species’ t 3FNPWF#4"BOE(FMBUJOQSPUFJOTUBCJMJ[FST immunoglobulin molecules. t 4QJO065™ columns to ensure optimal conditons for antibody clean Thiophilic adsorption has been used to purify other proteins 2 3 up including horseradish peroxidase , glutathione peroxidase , lactate 4 5 t 1FBSM™ IgG Puri!cation Resin for antibody clean up dehydrogenase and allergens . t 4VJUBCMFGPSYNM*H(4BNQMFT Supplied with protocols for IgG puri!cation, IgM puri!cation, IgY puri!cation and general protein puri!cation. APPLICATIONS The Thiophilic Adsorption kit is supplied with the thiophilic t 3FNPWF#4"(FMBUJOQSPUFJOTUBCJMJ[FSTUIBUJOUFSGFSFXJUI resin and all the necessary bu$ers for the rapid puri!cation of antibody labeling, fragmentation and isotyping experiments immunoglobulin G (IgG) antibodies. $BU Description Size 786-803 Pearl™ Antibody Clean Up 10 x 0.5ml samples

O S IgA PURIFICATION S OH """"""""""""""""" O O Immobilized Jacalin """"""""""""""""" Figure 6: Structure of thiophilic group on agarose beads. Jacalin, or Artocarpus integrifolia lectin, is a tetrameric two-chain FEATURES lectin with a molecular weight of 66kDa. Jacalin is a α-D-galactose t 1VSJGZXJEFSBOHFPGJNNVOPHMPCVMJONPMFDVMFT JODMVEJOH*H( *H. binding lectin puri!ed from jack-fruit (Artocarpus integrifolia) seeds. and IgY Applications include isolating IgA from human serum and colostrums, t )JHICJOEJOHDBQBDJUZ NHIVNBO*H(NMSFTJO isolating human plasma glycoproteins and histochemistry. Jacalin t #JOETDIJDLFOJNNVOPHMPCVMJO *H: also binds IgD. t (FOUMFFMVUJPODPOEJUJPOTJOWFSZMPXTBMUBOEOFBSOFBVUSBMQ) FEATURES t "EBQUBCMFUPPUIFSQSPUFJOT t #JOEJOH$BQBDJUZNHIVNBO*H"NMSFTJO t &OSJDINFOUBMUFSOBUJWFUPBNNPOJVNTVMGBUFQSFDJQJUBUJPO t -PBEJOHżNHKBDBMJONMPGSFTJO APPLICATIONS t 4VQQPSUDSPTTMJOLFEBHBSPTF t 1VSJGZJNNVOPHMPCVMJOT JODMVEJOH*H( *H.BOEDIJDLFO*H: APPLICATIONS REFERENCES t 1SFQBSJOH)VNBO*H"GSFFPGDPOUBNJOBUJOH*H( 1. Porath, J. et al (1984) In Physical Chemistry of Colloids and Macromolecules, Ed. Ranby, B. (Upsala, Sweden), p. 137 $BU Description Size 2. Chaga, G. et al (1992) Biomed. Chromatogr. 6:172 786-167 Immobilized Jacalin 2ml resin 3. Huang, K. et al (1994) Biol. Trace Elem. Res. 46:91 4. Kminkova, M. & Kucera, J. (1998) Prep. Biochem. Biotechnol. 28:313 5. Goubran-Bostros, H. et al (1998) J. Chromatogr. B. Biomed. Sci. Appl. 710:57 Jacalin, Lyophilized """"""""""""""""" $BU Description Size Artocarpus integrifolia lectin 786-266 Thiophilic Adsorption Kit 1 Kit 786-267 Thiophilic Resin 10ml resin Jacalin, or Artocarpus integrifolia lectin, is also available as a lyophilized protein. 786-268 Thiophilic Resin 100ml resin

$BU Description Size 786-473 Jacalin, lyophilized 10mg

8 For further details, visit www.GBiosciences.com Antibody Puri!cation AFFINITY COLUMN GENERATION Sulfhydryl Immobilization Kit for """"""""""""""""" Proteins Sulfhydryl Coupling Resin """"""""""""""""" """"""""""""""""" For generation of protein a#nity columns through Activated iodoacetyl group for binding free free sulfhydryls sulfhydryls The Sulfhydryl Immobilization Kit for Proteins is a complete kit The Sulfhydryl Coupling Resin is designed for the simple and designed for the simple and e"cient coupling of proteins to a solid FóDJFOUDPVQMJOHPGQFQUJEFTBOEQSPUFJOTUPBTPMJEBHBSPTF agarose support. The Sulfhydryl Coupling Resin Columns utilizes support through free sulfhydryl groups (-SH). The iodoacetyl groups iodoacetyl groups that speci!cally react with free sulfhydryls to form of the Sulfhydryl Coupling Resin speci!cally react with free sulfhydryls covalent, permanent thioether bonds (see !gure 7). The long spacer to form covalent, permanent thioether bonds (see !gure). The long arm reduces steric hindrance and ensures greater binding of proteins spacer arm reduces steric hindrance and ensures greater binding of and antibodies during a"nity puri!cation. proteins and antibodies during a"nity puri!cation. Proteins, including antibodies, must have free sulfhydryls The Sulfhydryl Coupling Resin is available as a resin slurry or for immobilization to the resin. A mild reducing agent, prealiquoted as !ve 2ml spin column format. 2-Mercaptoethylamine, is supplied to reduce the hinge region disul!de bonds of antibodies, while preserving the functionally crucial disul!de bonds between the heavy and light chains. The resulting columns can be used to study protein-protein interactions or for puri!cation, via a"nity chromatography. The columns, depending on the stability of the immobilized molecule, can be used several times without signi!cant loss of activity. FEATURES t (FOFSBUFTSFVTBCMF TQJOGPSNBUBóOJUZDPMVNOT t 4QFDJöDDPOKVHBUJPOUISPVHIGSFFTVMGIZESZMT t )JHI$BQBDJUZNHQSPUFJODPMVNO t 4VQQMJFEXJUINJMESFEVDJOHBHFOUGPSGSFFTVMGIZESZMTHFOFSBUJPO APPLICATIONS Figure 7: Sulfhydryl Coupling Resin scheme. t *NNPCJMJ[FQSPUFJOTUPQVSJGZJOUFSBDUJOHNPMFDVMFT FEATURES t *NNPCJMJ[FBOUJCPEJFTJOUIFDPSSFDUPSJFOUBUJPO t 4UBCMFDPVQMJOHPGQSPUFJOTBOEQFQUJEFT GPSNTDPWBMFOUUIJPFUIFS $BU Description Size bonds 786-804 Sulfhydryl Immobilization Kit for Proteins For 5 x 2ml columns t $PVQMFTNHQFQUJEFBOENHQSPUFJONMSFTJO APPLICATIONS Sulfhydryl Immobilization Kit for t 'PSUIFHFOFSBUJPOPGBóOJUZDPMVNOTGPSBOUJCPEZQVSJöDBUJPOBOE other a"nity chromatography Peptides """"""""""""""""" $BU Description Size For generation of peptide a#nity columns through 786-794 Sulfhydryl Coupling Resin 10ml resin free sulfhydryls 786-795 Sulfhydryl Coupling Resin 50ml resin 786-796 Sulfhydryl Coupling Resin 250ml resin Sulfhydryl Immobilization Kit for Peptides is designed for the 786-806 Sulfhydryl Coupling Resin 5 x 2ml columns simple and e"cient coupling of sulfhydryl-containing peptides to a solid agarose support. The Sulfhydryl Coupling Resin Columns utilizes iodoacetyl groups that speci!cally react with free sulfhydryls to form covalent, permanent thioether bonds (see !gure 7). The long spacer arm reduces steric hindrance and ensures greater binding of proteins and antibodies during a"nity puri!cation. Peptides must have free sulfhydryls for immobilization to the resin. The supplied Protein-S-S-Reductant™ reducing agent e"ciently reduces disul!de bonds and does not interfere with the iodoacetyl coupling reaction. Protein-S-S-Reductant™ o$ers the advantage that it does not require removal before peptide immobilization. The resulting columns can be used for the puri!cation of antibodies that have been raised against the speci!c peptide. The columns, depending on peptide stability, can be used several times. FEATURES t (FOFSBUFTSFVTBCMF TQJOGPSNBUBóOJUZDPMVNOT t 4QFDJöDDPOKVHBUJPOUISPVHIGSFFTVMGIZESZMT t )JHI$BQBDJUZNHQFQUJEFDPMVNO APPLICATIONS t *NNPCJMJ[FQFQUJEFTGPSBOUJCPEZQVSJöDBUJPO

$BU Description Size 786-805 Sulfhydryl Immobilization Kit for Peptides For 5 x 2ml columns For further details, visit www.GBiosciences.com 9 Antibody Puri!cation Amine Coupling Resin CDI Amine Reactive Resin """"""""""""""""" """"""""""""""""" The amine reactive HOOK™"DUJWBUFE"HBSPTFJTBHBSPTF (#JPTDJFODFT$%*"NJOF3FBDUJWF"HBSPTFDPOTJTUTPGDSPTT that has been activated to generate reactive aldehyde groups. The linked agarose activated with CDI (1,1’-carbonyl diimidazole) to form aldehyde groups of the agarose react spontaneously with primary reactive imidazole carbamates. amines, located at the N-terminus of proteins or in lysine residues, to The activation of the resin occurs in solvent and to maintain its form intermediate Schi$ Base complexes. These, in turn, are selectively activity the resin is supplied in acetone to prevent hydrolysis. Upon reduced by reductive amination to form stable amine linkages reaction of the resin with primary amine containing molecules, between the agarose and the ligand. i.e. proteins, in basic (pH8.5-10) aqueous bu$ers the imidazole carbamates lose the imidazole group and form carbamate linkages. CDI Amine Reactive Agarose is ideal for immobilizing peptides, small organic molecules and certain proteins and reactions can occur in organic solvent making it ideal for water-insoluble ligands.

Figure 8: Scheme for the coupling of proteins to HOOK™ Activated Agarose (Amine Reactive). The amine reactive HOOK™ Activated agarose is also supplied in a complete kit for the generation of 5 x 2ml resins. The kit is supplied Figure 9: Scheme for the coupling of proteins to CDI Amine Reactive with all the necessary reagents and columns. Agarose. FEATURES The amine reactive HOOK™ Activated agarose is also supplied in a t #JOEJOHDBQBDJUZNHQSPUFJONMSFTJO complete kit for the generation of 5 x 2ml resins. The kit is supplied t DSPTTMJOLFEBHBSPTF with all the necessary reagents and columns. APPLICATIONS FEATURES t $PVQMJOHPGQSPUFJOTBOEQFQUJEFTUPBHBSPTFCFBET t 1SPWFODPVQMJOHDIFNJTUSZ t 4VJUBCMFGPSBOUJCPEZQVSJöDBUJPO t &BTZUPVTF OPTFDPOEBSZDPVQMJOHBHFOUTSFRVJSFE Cat Description Size t 4UBCMFMJOLBHFT t $PVQMFJOJOPSHBOJDCVòFSTGPSJOTPMVCMFNPMFDVMFT 786-066 HOOK™ Activated Agarose (Amine Reactive) 10ml resin HOOK™ Activated Agarose Coupling Kit For 5 x 2ml APPLICATIONS 786-063 (Amine Reactive) columns t $PVQMFQSPUFJOTBOEQFQUJEFT t $PVQMFQSJNBSZBNJOFDPOUBJOJOHMJHBOET

Cat Description Size 786-404 CDI Amine Reactive Resin 10ml resin

10 For further details, visit www.GBiosciences.com Antibody Puri!cation Carboxyl Coupling Resin SDC™ (Steroid/Drug/Compound) """"""""""""""""" $POTJTUTPGDSPTTMJOLFEBHBSPTFXJUIDPWBMFOUMJOLFE Immobilization diaminodipropylamine (DADPA) to generate a free primary amine at """"""""""""""""" the end of a long spacer arm. Designed for the immobilization of steroids, drugs and chemical compounds that lack primary amines, sulfhydryls, carbonyls and other common coupling groups to a solid-phase agarose support for the use in a"nity puri!cation. The kit uses Immobilized DADPA (diaminodipropylamine) resin to bind steroids, drugs and chemicals through their active hydrogens. The coupling uses the Mannich reaction, which is described as the condensation of formaldehyde with ammonia, in the form of its salt, and another compound containing an active hydrogen. The SDC™ Immobilization kit replaces the ammonia with the primary amine on the DADPA and the active hydrogen is supplied by the steroid, drug or chemical to be coupled. Ideal for the generation of !ve 2ml a"nity columns. Figure 10: Carboxyl Coupling Resin scheme. Molecules, including proteins and peptides, are covalently coupled to the free primary amines, and the stable columns are ideal for a"nity puri!cation of antibodies and other interacting partners. Molecules can be coupled to the free amine by numerous amine- reactive methods; however the use of the carbodiimide EDC allows coupling of free carboxyl groups. The resulting amide bond is highly stable and greatly reduces the chance of leaching of the a"nity tag. The long spacer arm reduces steric hindrance and ensures greater binding of proteins and antibodies during a"nity puri!cation. FEATURES t *NNPCJMJ[FE%"%1" EJBNJOPEJQSPQZMBNJOF t DSPTTMJOLFEBHBSPTF Figure 11: Active hydrogen containing compounds. t -POHTQBDFSBSNUPMJNJUTUFSJDIJOESBODF t $PVQMFDBSCPYZMHSPVQT APPLICATIONS t $PVQMFQFQUJEFTGPSBOUJCPEZQVSJöDBUJPO t $PVQMFQFQUJEFTBOEQSPUFJOTUPQVSJGZJOUFSBDUJOHNPMFDVMFT

Cat Description Size Carboxyl Coupling Resin (Immobilized DADPA 786-797 25ml resin (Diaminodipropylamine))

Figure 12: SDC™ (Steroid/ Drug/ Compound) Immobilization scheme.

FEATURES t 6TFT*NNPCJMJ[FE%"%1" EJBNJOPEJQSPQZMBNJOF SFTJO t 4UBCMF DPWBMFOUMJOLBHF APPLICATIONS t *NNPCJMJ[BUJPOPGESVHT TUFSPJETBOETNBMMNFUBCPMJUFTUISPVHI active hydrogens t *EFBMGPSDPNQPVOETMBDLJOHQSJNBSZBNJOFT TVMGIZESZMT DBSCPOZMT and other common coupling groups

Cat Description Size 786-271 SDC™ (Steroid/Drug/Compound) Immobilization 5 reactions For further details, visit www.GBiosciences.com 11 Antibody Puri!cation COATED 96#WELL PLATES Well-Coated™ Protein L """"""""""""""""" """"""""""""""""" Well-Coated™ plates are available as single 96-well plates or as 12 x Bind kappa light chains of immunoglobulins 8-well strips in a 96-well holder. The plates are supplied as clear, white Designed to bind the kappa light chains of immunoglobulins and black plates for colorimetric, chemiluminescence and #uorescent without interfering with the antigen binding site. Well-Coated™ detection systems respectively. Protein L plates bind a greater range of immunoglobulin classes and ™ subclasses compared to Protein A, G and A/G. Protein L will bind to all Well-Coated Protein A, Protein G & classes of IgG, including IgG, IgM, IgA, IgE and IgD, and binds to single Protein A/G chain variable fragments (scFv and Fab fragments). """""""""""""""" The plates are for single antibody assays and are not suitable for Bind constant (Fc) domain of antibodies multiple assays (sandwich ELISAs) as the !rst antibody will not block all IgG binding sites. The wells are coated to a 100µl depth and are Designed to bind the constant (Fc) region of immunoglobulins supplied pre-blocked. Clear, white and black plates are o$ered. ensuring that the antigen binding domain of the antibody is FEATURES orientated away from the plate, o$ering maximum exposure of the t 3FUBJOTBOUJCPEZBDUJWJUZ binding site. Protein A-G contains 4 binding sites from protein A and t #JOETUPBMMDMBTTFTPG*H( JODMVEJOH*H( *H. *H" *H&BOE*H% 2 from protein G o$ering maximum range of speci!city and binding t 3FEVDFEOPOTQFDJöDCJOEJOHBTQMBUFTBSFQSFCMPDLFE capacity. The immunoglobulin orientation improves the antibody capacity compared to plates that are coated directly with antibodies. TECHNICAL INFORMATION The plates are for single antibody assays and are not suitable for t 0OMZCJOETLBQQB* ***BOE*7JOIVNBOBOELBQQB*JONPVTF multiple assays (sandwich ELISAs) as the !rst antibody will not block t .BZCFTQFDJöDGPSDFSUBJOLBQQBTVCHSPVQTJOPUIFSTQFDJFT all IgG binding sites and therefore false positives will occur with the t #JOETTD'WXJUIPVUJOUFSGFSJOHXJUIBOUJHFOCJOEJOH second antibody. The wells are coated to a 100µl depth and are t )BTXFBLCJOEJOHBóOJUZGPSSBCCJUJNNVOPHMPCVMJOT supplied pre-blocked. Clear, white and black plates are available. t /PCJOEJOHBóOJUZGPSCPWJOF HPBUPSTIFFQJNNVOPHMPCVMJOT See table 1 for antibody binding a"nities of Protein A, Protein G t /PCJOEJOHBóOJUZGPSMBNCEBMJHIUDIBJOT and Protein A/G $BU Description Size FEATURES 786-737 Well-Coated™ Protein L, 8-well strip plate, Clear 5 Plates t 1SPUFJO"(IBTIJHIFTUTQFDJöDJUZBOEDBQBDJUZ 786-776 Well-Coated™ Protein L, 96 well plate, Black 5 Plates t 3FUBJOTBOUJCPEZBDUJWJUZPSJFOUTBOUJCPEZGPSNBYJNVNCJOEJOH 786-736 Well-Coated™ Protein L, 96 well plate, Clear 5 Plates t 3FEVDFOPOTQFDJöDCJOEJOH 786-777 Well-Coated™ Protein L, 96 well plate, White 5 Plates t #JOET_QNPMSBCCJU*H(XFMM $BU Description Size Well-Coated™ Antibody 786-731 Well-Coated™ Protein A, 8-well strip plate, Clear 5 Plates """"""""""""""""" 786-770 Well-Coated™ Protein A, 96 well plate, Black 5 Plates Bind mouse or rabbit IgG antibodies 786-730 Well-Coated™ Protein A, 96 well plate, Clear 5 Plates Designed to speci!cally bind either mouse or rabbit IgG making 786-771 Well-Coated™ Protein A, 96 well plate, White 5 Plates them suitable for binding assays using low quantities of antibodies 786-733 Well-Coated™ Protein G, 8-well strip plate, Clear 5 Plates or antibodies that denature on direct binding to polystyrene plates. 786-774 Well-Coated™ Protein G, 96 well plate, Black 5 Plates Another advantage is that the speci!city to IgG means puri!ed 786-732 Well-Coated™ Protein G, 96 well plate, Clear 5 Plates antibodies are not essential. 786-775 Well-Coated™ Protein G, 96 well plate, White 5 Plates Suitable for direct, indirect, competitive and sandwich assays. The wells are coated to a 100µl depth and are supplied pre-blocked. Clear, 786-735 Well-Coated™ Protein A/G, 8-well strip plate, Clear 5 Plates white and black plates are o$ered. 786-772 Well-Coated™ Protein A/G, 96 well plate, Black 5 Plates 786-734 Well-Coated™ Protein A/G, 96 well plate, Clear 5 Plates FEATURES 786-773 Well-Coated™ Protein A/G, 96 well plate, White 5 Plates t #JOET_QNPMNPVTF*H(XFMMPS_QNPMSBCCJU*H(XFMM t 1SFWFOUTEFOBUVSBUJPOPGBOUJCPEJFTVOMJLFEJSFDUCJOEJOH t 4QFDJFTTQFDJöDCJOEJOH $BU Description Size 786-739 Well-Coated™ Antibody (goat α-mouse), 8-well strip, Clear 5 Plates 786-758 Well-Coated™ Antibody (goat α-mouse), 96 well, Black 5 Plates 786-738 Well-Coated™ Antibody (goat α-mouse), 96 well, Clear 5 Plates 786-759 Well-Coated™ Antibody (goat α-mouse), 96 well, White 5 Plates 786-741 Well-Coated™ Antibody (goat α-rabbit), 8-well strip, Clear 5 Plates 786-760 Well-Coated™ Antibody (goat α-rabbit), 96 well, Black 5 Plates 786-740 Well-Coated™ Antibody (goat α-rabbit), 96 well, Clear 5 Plates 786-761 Well-Coated™ Antibody (goat α-rabbit), 96 well, White 5 Plates

12 For further details, visit www.GBiosciences.com Antibody Fragmentation A large selection of reagents and kits for the generation of Fc, Fab and F(ab) fragments from IgG antibodies. Utilizes optimized, Immobilized Ficin 2 """"""""""""""""" immobilized papain, pepsin and !cin proteases.

FICIN

Immobilized Papain -S-S- -S-S- -S-S- -S-S-

""""""""""""""""" -S-S- -S-S- -S-S- -S-S- 1mM cysteine

PAPAIN F(ab’)2

10mM cysteine

-S-S-

-S-S- -S-S-

-S-S- -S-S- -S-S- IgG -S-S- -S-S- -S-S- 2 Fab Figure 15: Digestion of Immunglobulin G with Ficin.

-S-S- Ficin (or Ficain) is a cysteine protease enzyme (EC 3.4.22.3) isolated from !g latex is that has the endopeptidase activity to IgG Fc 2 Fab cleave immunoglobulin G molecules in the hinge reason. Ficin is Figure 13: Digestion of Immunglobulin G with Papain. typically used to cleave mouse IgG1 as this are di"cult to cleave with "DZTUFJOFQSPUFBTFFO[ZNF &$ JNNPCJMJ[FEPO papain and pepsin. In the presence of 1mM or 10mM cysteine, !cin agarose, cleaves immunoglobulin G antibody molecules in the hinge generates F(ab)2 and Fab fragments respectively. Immobilized Ficin

SFHJPO HFOFSBUJOHUISFF_L%BGSBHNFOUTUXP'BCEPNBJOTBOE is a convenient reagent for producing Fab and F(ab)2 fragments as it a Fc domain. The papain-digested antibody is unable to promote avoids the need to remove the !cin enzyme after digestion. agglutination, precipitation, opsonization, and lysis. FEATURES

FEATURES t (FOFSBUF'BCBOE' BCA 2 fragments t (FOFSBUF'DBOE'BCGSPN*H( t 'PSEJHFTUJPOPGNPVTF*H( t &MJNJOBUFTDPOUBNJOBUJPOXJUIQBQBJOFO[ZNF t &MJNJOBUFTDPOUBNJOBUJPOCZ'JDJO t $BOCFVTFEJOWJSUVBMMZBMMTDFOBSJPTVTJOHGSFFQBQBJO $BU Description Size $BU Description Size 786-793 Immobilized Ficin 5ml Resin 786-790 Immobilized Papain 5ml Resin Fab Fragmentation Immobilized Pepsin """"""""""""""""" """"""""""""""""" Designed for the generation and isolation of Fab fragments from IgG molecules. The kits utilize our Immobilized Papain resin. Papain is a cysteine protease enzyme (EC 3.4.22.2) that has the endopeptidase

-S-S- -S-S- -S-S- activity to cleave immunoglobulin G molecules in the hinge reason. -S-S-

-S-S- -S-S- -S-S- 5IJTDMFBWBHFSFTVMUTJOUIFHFOFSBUJPOPGUISFF_L%BGSBHNFOUT -S-S- two Fab domains and a Fc domain. The papain-digested antibody PEPSIN PEPSIN

F(ab’)2 is unable to promote agglutination, precipitation, opsonization, and lysis. Immobilized Papain o$ers the advantage of generating Fab and IgG Fc fragments without the need to remove the papain enzyme after Figure 14: Digestion of Immunglobulin G with Pepsin. digestion. Following papain digestion the Fab fragments are separated from "QSPUFPMZUJDFO[ZNFJNNPCJMJ[FEPOBHBSPTFUIBUJTSPVUJOFMZ undigested IgG and the Fc region with the supplied Protein A Spin used for the generation of F(ab’) fragments from immunoglobulin G 2 Column. The Protein A Resin binds the IgG and Fc molecules and the (IgG). The pepsin has the ability to cleave the heavy chains near the Fab are rapidly collected due to the spin-format design. hinge region. One or more of the disul!de bonds that join the heavy In addition, SpinOUT™ GT-600 desalting columns are supplied to chains in the hinge region are preserved, so the two Fab regions of ensure the initial antibody sample is in the optimal condition for Fab the antibody remain joined together, yielding a divalent molecule Fragmentation. (containing two antibody binding sites), hence the designation The Fab Fragmentation kit is optimized for mouse, rabbit and F(ab) . The light chains remain intact and attached to the heavy chain, 2 human IgG, using 0.25-4mg/ 0.5ml sample. whereas the Fc fragment is digested into small peptides. The Fab Fragmentation (Micro) kit is optimized for mouse, rabbit The Immobilized Pepsin o$ers the distinct advantage of and human IgG, using 25-250µg/ 125µl sample. eliminating enzyme contamination of the F(ab)2 fragments. FEATURES PAPAIN -S-S-

t (FOFSBUF' BC fragments -S-S-

-S-S- 2 -S-S-

t &MJNJOBUFDPOUBNJOBUJOHQFQTJOFO[ZNF -S-S- -S-S- -S-S- t $BOCFVTFEJOWJSUVBMMZBMMTDFOBSJPTVTJOHGSFFQFQTJO -S-S- Immobilized + Protein A $BU Description Size Papain Resin Fc -S-S- -S-S- 786-791 Immobilized Pepsin 5ml Resin 2 Fab 2 Fab IgG Figure 16: Fab Fragmentation scheme.

For further details, visit www.GBiosciences.com 13 Antibody Fragmentation FEATURES Fab & F(ab) Fragmentation of t *NNPCJMJ[FE1BQBJOFOTVSFTOPFO[ZNFDPOUBNJOBUJPO 2 t 0QUJNJ[FEGPSIVNBO NPVTF BOESBCCJU*H( Mouse IgG1 t 3FBEZUPVTF'BCGSBHNFOUT XJUIFOIBODFEZJFMEBOEQVSJUZ """"""""""""""""" t 4QJOGPSNBUGPSSBQJEQVSJöDBUJPO Designed for the generation and isolation of Fab and F(ab)2 t $POUBJOTBMMFTTFOUJBMSFBHFOUT fragments from mouse IgG1 molecules. t 5XPDPOWFOJFOULJUTJ[FTBWBJMBCMF The kit utilizes our Immobilized Ficin resin. Ficin (or Ficain)

* For mouse IgG1 fragmentation we recommend our Fab & F(ab)2 _ %B JTBDZTUFJOFQSPUFBTFFO[ZNF &$ JTPMBUFE

Fragmentation of Mouse IgG1 kits from !g latex is that has the endopeptidase activity to cleave immunoglobulin G molecules in the hinge reason. Ficin has an $BU Description Size e$ective range of pH4-9.5 with an optimal pH of 6.5 and cleaves 786-272 Fab Preparation Kit 10 reactions bonds that involve uncharged or aromatic amino acids. 786-273 Fab Preparation Kit (Micro) 10 reactions Ficin is typically used to cleave mouse IgG1 as this are di"cult to cleave with papain and pepsin. In the presence of 1-4mM or 10- 20mM cysteine, !cin generates F(ab) and Fab fragments respectively. F(ab)2 Fragmentation 2 """"""""""""""""" Immobilized Ficin is a convenient reagent for producing Fab andF(ab)2 fragments as it avoids the need to remove the !cin enzyme after The F(ab)2 Fragmentation kits are designed for the generation and digestion. isolation of F(ab)2 fragments from IgG molecules. The kit utilizes our Immobilized Pepsin resin. Pepsin is a proteolytic Following !cin digestion the fragments are separated from undigested IgG and the large Fc fragments with the supplied Protein A enzyme that is routinely used for the generation of F(ab)2 fragments from immunoglobulin G (IgG). The pepsin has the ability to cleave Spin Column. The Protein A Resin binds the IgG and Fc molecules and

the heavy chains near the hinge region. One or more of the disul!de the Fab or F(ab)2 are rapidly collected due to the spin-format design. bonds that join the heavy chains in the hinge region are preserved, so In addition, SpinOUT™ GT-600 desalting columns are supplied the two Fab regions of the antibody remain joined together, yielding a to ensure the initial antibody sample is in the optimal condition for divalent molecule (containing two antibody binding sites), hence the fragmentation. The Fab &F(ab) Fragmentation of Mouse IgG kit is optimized for designation F(ab)2. The light chains remain intact and attached to the 2 1 heavy chain, whereas the Fc fragment is digested into small peptides. mouse IgG1, using 0.25-4mg/ 0.5ml sample.

The Immobilized Pepsin o$ers the distinct advantage of eliminating The Fab & F(ab)2 Fragmentation of Mouse IgG1 Micro kit is optimized for mouse IgG , using 25-250µg/ 125µl sample. enzyme contamination of the F(ab)2 fragments. 1

Following pepsin digestion the F(ab)2 fragments are separated from undigested IgG and the large Fc fragments with the supplied -S-S- Protein A Spin Column. The Protein A Resin binds the IgG and Fc -S-S- -S-S- -S-S- FICIN -S-S- -S-S- F(ab’) F(ab’)2 molecules and the F(ab)2 are rapidly collected due to the spin-format 2

-S-S- design. -S-S-

™ -S-S- -S-S- -S-S- In addition, SpinOUT GT-600 desalting columns are supplied to -S-S- 1mM cysteine Immobilized ensure the initial antibody sample is in the optimal condition for F(ab) Ficin 2 20mM cysteine + Protein A Fragmentation. Resin or

The F(ab)2 Fragmentation kit is optimized for mouse, rabbit and

-S-S- IgG -S-S- human IgG, using 0.25-4mg/ 0.5ml sample. 2 Fab 2 Fab The F(ab)2 Fragmentation (Micro) kit is optimized for mouse, rabbit and human IgG, using 25-250µg/ 0.125ml sample. -S-S- -S-S-

Figure 18: Fab & F(ab)2 Fragmentation of Mouse IgG1 scheme.

-S-S- -S-S- -S-S- Immobilized FEATURES -S-S- -S-S- -S-S- -S-S- -S-S- Pepsin -S-S- -S-S- t *NNPCJMJ[FE'JDJOFOTVSFTOPFO[ZNFDPOUBNJOBUJPOPGEJHFTUJPOT + Protein A Resin t 0QUJNJ[FEGPSNPVTF*H(1 -S-S- -S-S- PEPSIN PEPSIN t 3FTVMUTJOSFBEZUPVTF'BCPS' BC GSBHNFOUT XJUIFOIBODFE yield and purity F(ab) F(ab) IgG 2 2 t 4QJOGPSNBUGPSSBQJEQVSJöDBUJPO Figure 17: F(ab)2 Fragmentation scheme. t $POUBJOTBMMFTTFOUJBMSFBHFOUT t 5XPDPOWFOJFOULJUTJ[FTBWBJMBCMF FEATURES t *NNPCJMJ[FE1FQTJOFOTVSFTOPFO[ZNFDPOUBNJOBUJPO $BU Description Size

t 0QUJNJ[FEGPSIVNBO NPVTF BOESBCCJU*H( 786-276 Mouse IgG1 Fab & F(ab)2 Preparation Kit 10 reactions

t 3FBEZUPVTF' BC 2 fragments, with enhanced yield and purity 786-277 Mouse IgG1 Fab & F(ab)2 Preparation Kit (Micro) 10 reactions t 4QJOGPSNBUGPSSBQJEQVSJöDBUJPO t $POUBJOTBMMFTTFOUJBMSFBHFOUT t 5XPDPOWFOJFOULJUTJ[FTBWBJMBCMF

* For mouse IgG1 fragmentation we recommend our Fab & F(ab)2

Fragmentation of Mouse IgG1 kits. $BU Description Size

786-274 F(ab)2 Preparation Kit 10 reactions

786-275 F(ab)2 Preparation Kit (Micro) 10 reactions

14 For further details, visit www.GBiosciences.com Antibody Labeling FLUORESCENT DYE LABELING HOOK™ Dye Labeling Kit (FITC) """"""""""""""""" """"""""""""""""" The labeling of proteins with #uorescent dyes has become an important research tool in many !elds. Two kits are o$ered for labeling virtually any protein, particularly antibodies, with either a rhodamine or #uorescein based dye. HOOK™ Dye Labeling Kit (5/6) TAMRA-SE (Rhodamine) """""""""""""""""

Figure 21: Structure of $uorescein isothiocyanate. FITC (#uorescein isothiocyanate) is a commonly used #uorescent label for proteins, as it contains the groups required for conjugating to amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups of proteins. FITC has a molecular weight of 389, and excitation and emission wavelengths of 494nm and 520nm, respectively, therefore emitting green visible light. ™ Figure 19: Structure of (5/6) TAMRA-SE. This kit utilizes SpinOUT columns for the rapid puri!cation of dye labeled proteins. (5/6) TAMRA-SE (5-(and-6)- Carboxytetramethylrhodamine succinimidyl ester, mixed isomers) is based on tetramethylrhodamine, one of the most common #uorophores used in the labeling of peptides, proteins, nucleic acids and nucleotides. (5/6) TAMRA absorbs green visible light at 546nm and emits an orange-red visible light at a maximum emission of 575nm. The NHS ester group provides the simplest and most commonly used group for labeling proteins. The succinimidyl ester group reacts with primary amines in lysine side chains and N-terminal amines forming a stable, covalent amide bond. This kit utilizes SpinOUT™ columns for the rapid puri!cation of dye labeled proteins. Figure 22: Visualization of FITC Labeled Casein. Lane 1: 1μg FITC labeled casein, Lane 2-3: 1μg FITC-Casein digested with 0.2μg or 0.1μg Trypsin. Samples were resolved on a 4-20% SDS polyacrylamide gel.

FEATURES t %ZFQSFXFJHIFEBOETVQQMJFEJOTJOHMFVTF0OF2VBOU™ vials t 4VJUBCMFGPSNPTUQSPUFJOT ™ Figure 20: Visualization of TAMRA labeled BSA. 1μg (5/6) TAMRA-SE labeled t 6UJMJ[FT4QJO065 desalting columns to isolate labeled protein BSA was resolved on a 4-20% SDS polyacrylamide gel. APPLICATIONS t -BCFMJOHPGBHSFFOøVPSFTDFOUEZFUPQSPUFJOTBOEQFQUJEFT FEATURES t 4VJUBCMFGPSBOUJCPEZMBCFMJOH t %ZFQSFXFJHIFEBOETVQQMJFEJOTJOHMFVTF0OF2VBOU™ vials t 4VJUBCMFGPSNPTUQSPUFJOT $BU Description Size t 6UJMJ[FT4QJO065™ desalting columns to isolate labeled protein 786-141 HOOK™ FITC Labeling Kit 1 kit APPLICATIONS t -BCFMJOHPGQSPUFJOT QFQUJEFTBOEOVDMFJDBDJETXJUIBSFE OneQuant™ Fluorescent Reagents #uorescent dye """"""""""""""""" t 4VJUBCMFGPSBOUJCPEZMBCFMJOH Both the #uorescent reagents (FITC and (5/6) TAMRA) are available in our OneQuant™ format. $BU Description Size The OneQuant™ format prevents loss of reagent due to repeated ™ 786-142 HOOK (5/6) TAMRA-SE (Rhodamine) Labeling Kit 1 kit weighing. Each vial also limits exposure to light.

$BU Description Size 786-079 OneQuant™ TAMRA 8 x 0.5mg 786-080 OneQuant™ FITC 8 x 1mg

For further details, visit www.GBiosciences.com 15 Antibody Labeling BIOTIN LABELING COUPLING FACTORS """"""""""""""""" """"""""""""""""" A non-radioactive labeling system Spacer Arms """"""""""""""""" The biotin-binding domain in avidin/ streptavidin molecules are buried 9Å below the surface and hence, the presence of bulky groups in the vicinity of the biotin-binding site may create steric hindrances and reduce the binding e"ciency and the sensitivity of detection methods. Greater binding capacity can be realized by using biotin derivatives that have large spacer arms. Extended spacer arms a$ord the ability to overcome steric hindrances and bind deep within the binding sites of the avidin/ streptavidin molecules. Figure 23: Structure of Biotin. #JPUJO B%BMUPOWJUBNJO 7JUBNJO) NPMFDVMF FYIJCJUTBO Solubility 15 -1 """"""""""""""""" extraordinary binding a"nity for avidin (Ka=10 M ) and streptavidin. Solubility of the HOOK™-Biotin Reagents varies greatly, with some Biotin and avidin interaction is rapid and once the bond is established being only soluble in organic solvents, i.e. DMSO and DMF. it can survive up to 3M guanidine-hydrochloride and extremes of pH. Biotin-avidin bonds can only be reversed by denaturing the avidin protein molecule with 8M guanidine-hydrochloride at pH1.5 Membrane Permeability or by autoclaving. The biotinylated molecules are e"ciently probed """"""""""""""""" Membrane permeability has become of great interest in studies with avidin or streptavidin conjugated to reporter molecules, such as of cell surface proteins and therefore membrane tra"cking and peroxidases or phosphatases. The use of biotin for non-radioactive cell signaling. The HOOK™ Biotin Reagents that are not membrane labeling of proteins and nucleic acids has now become an increasingly permeable are excellent candidates for labeling membrane surface popular technique in life science research. Avidin is a glycoprotein proteins. XJUIBQQSPYJNBUFMZPGJUTUPUBMNBTTDPNJOHGSPNDBSCPIZESBUFT Avidin has a molecular weight of 67kDa and contains four identical 128 amino acid subunits that each have a single biotin binding Reversibility domain. Avidin is a basic protein with an isoelectric pH of 10-10.5 """"""""""""""""" Biotin tags are often used for protein puri!cation, however with and is readily soluble in aqueous bu$ers containing a wide range of the biotin:avidin binding a"nity being one of the strongest known salt, pH, temperature and other laboratory agents. This wide range it is often di"cult to release the protein from the avidin. In fact, of tolerance makes avidin suitable for a wide variety of analytical 8M guanidine at pH1.5 is often used, which has severe detrimental applications. Streptavidin is a tetrameric protein and in many respects e$ects on the protein of interest. Several HOOK™ Biotin Reagents have is similar to avidin except that it has no carbohydrate and has a slightly disul!de bonds that can be reduced to release the protein of interest lower molecular weight of about 60kDa. The solubility of streptavidin under mild conditions and other HOOK™ Biotin Reagents can be (isoelectric pH5) in aqueous bu$er is much lower than avidin, but the removed from the protein with changes in pH. binding of streptavidin to biotin is similar to that of avidin. Several factors must be considered when coupling a biotin reagent to a protein to ensure a successful reaction. The primary consideration Reactive Groups is the selection of the biotinylation reagent itself. A wide range """"""""""""""""" of biotin reagents are o$ered that have variations in their reactive The reagents o$ered have numerous reactive groups that groups, spacer arm lengths, solubility, membrane permeability and can couple to amines, sulfhydryls, carboxyls and carbohydrates. reversibility. All these factors must be considered and are dependent Conjugation of biotin reagents to proteins and other molecules on your protein/peptide. generally does not have adverse e$ects on the biological properties of the target molecules, unless biotin reagents are conjugating to or modifying active residues or sites of the protein. Due to this, it is important to !nd an appropriate biotin reagent and optimal biotin conjugation e"ciency for maintaining the functional properties of the target molecules. The conjugation e"ciency of the reactions is dependent on the reaction groups and the bu$ers used for the reactions as many coupling reactions are sensitive to pH and chemical composition. The following section highlights the key features of the coupling reactions and important bu$er information. Based on the target reactive groups, biotin reagents can be divided into amine reactive, sulfhydryl reactive, carbohydrate reactive, and carboxyl reactive. Photoreactive biotin reagents react non-speci!cally upon exposure UP67MJHIUBOEBSFVTFEXIFOOPBQQSPQSJBUFSFBDUJWFUBSHFUJT available on the molecules.

16 For further details, visit www.GBiosciences.com Antibody Labeling Biotin Selection Guide """"""""""""""""" t Reactive Group: Determines the location of the biotin moiety t .FNCSBOF1FSNFBCJMJUZ For cell surface labeling select non membrane permeable reagents t Cleavable: For easy removal from immobilized avidin or streptavidin during puri!cation t Reversible: An alternative to cleavable reagents are reversible reagents t Steric Hinderance: Bulky groups around the binding site may require reagents with longer spacer arms

Reactive olecular H oluble ™ pacer $BU HOOK Biotin Reagent Size M 8FJHIU S "SN ¯ Group .FNCSBOF 1FSNFBCMF 8BUFS S Cleavable/ Reversible Reaction p E#JPUJO WJUBNJO) BG-00 500mg 244.32 0

AMINE REACTIVE REAGENTS HOOK™-NHS-Biotin BG-01 50mg 341.38 13.5 NHS-ester YES NO NO 7-9 786-083 8 x 2mg

HOOK™-NHS-LC-Biotin

BG-02 50mg 454.54 22.4 NHS-ester YES NO NO 7-9

HOOK™-NHS-LC-LC-Biotin

BG-03 50mg 567.70 30.5 NHS-ester YES NO NO 7-9

HOOK™-NHS-SS-Biotin

BG-04 50mg 504.65 24.3 NHS-ester YES NO YES 7-9

™ HOOK NHS-dPEG4 -Biotin BG-05 O 50mg NH HN

O 588.67 29 NHS-ester NO YES NO 7-9 H O O N O O O S N O 786-700 O 8 x 1mg O

HOOK™-sulfo-NHS-Biotin BG-06 50mg sulfo-NHS 443.43 13.5 NO YES NO 7-9 ester 786-698 8 x 1mg

HOOK™-sulfo-NHS-LC-Biotin BG-07 50mg sulfo-NHS 556.59 22.4 NO YES NO 7-9 ester 786-084 8 x 1mg

HOOK™-sulfo-NHS-LC-LC-Biotin sulfo-NHS BG-08 50mg 669.75 30.5 NO YES NO 7-9 ester

HOOK™-sulfo-NHS-SS-Biotin BG-09 50mg sulfo-NHS 606.69 24.3 NO YES YES 7-9 ester 786-699 8 x 1mg

HOOK™-PFP-Biotin

Penta#uoro- BG-10 50mg 410.36 9.6 YES NO NO 7-9 phenyl ester

For further details, visit www.GBiosciences.com 17 Antibody Labeling

Reactive olecular H oluble ™ pacer $BU HOOK Biotin Reagent Size M 8FJHIU S "SN ¯ Group .FNCSBOF 1FSNFBCMF 8BUFS S Cleavable/ Reversible Reaction p SULFHYDRYL REACTIVE REAGENTS ™ HOOK -PEG-Iodoacetyl-Biotin

BG-11 50mg 542.43 24.7 Iodoacetyl NO YES NO 7.5-8.5

HOOK™-Iodoacetyl-LC-Biotin BG-12 50mg 510.43 27.1 Iodoacetyl YES NO NO 7.5-8.5

786-085 8 x 2mg

HOOK™-Biotin-PDA BG-13 50mg 412.60 21.1 Pyridyldithiol YES NO YES 6-9

HOOK™-Biotin-BMCC

BG-14 50mg 533.68 32.6 Maleimide NO NO NO 6.5-7.5

CARBOXYL REACTIVE REAGENTS HOOK™-#JPUJO1FOUZMBNJOF

BG-15 50mg 328.47 18.9 Amine NO YES NO 4-6

™ HOOK -Biotin-PEG"NJOF

BG-16 50mg 374.50 20.4 Amine NO YES NO 4-6

™ HOOK -Biotin-PEG"NJOF

BG-17 50mg 418.55 22.9 Amine NO YES NO 4-6

CARBOHYDRATE REACTIVE REAGENTS HOOK™-Biotin-Hydrazide

BG-18 50mg 258.34 15.7 Hydrazide YES NO NO 4-6

HOOK™-Biotin-LC-Hydrazide

BG-19 50mg 371.50 24.7 Hydrazide YES NO NO 4-6

PHOTOREACTIVE REAGENTS HOOK™-Psoralen-PEO-Biotin

BG-20 5mg 688.79 36.9 Psoralen NO YES NO 4-6

Table 3: Biotin Selection Guide.

18 For further details, visit www.GBiosciences.com Antibody Labeling ™ 0 /) OneQuant Biotin Reagents Sulfhydryl Reactive )/ 0

) 0 """"""""""""""""" * / 4 / ™ ) 0 )/ 0 /) Several of the more commonly used HOOK Biotin reagents are ) / / ™ ) 4 available in our OneQuant format. 0 EDC 0 Carboxyl Reactive ™ 0 )/ 0 The OneQuant format prevents loss of reagent due to repeated /) / 0 ™ /) 0 )/ weighing as each vial contains only 1-2mg HOOK Biotin Reagent. 4

) Amine Reactive 0 / 0 4 )/ $BU Description Size 0 Carbohydrate Reactive 786-083 OneQuant™ HOOK™ NHS-Biotin 8 x 2mg Sodium meta-periodate 786-084 OneQuant™ HOOK™ Sulfo-NHS-LC-Biotin 8 x 1mg 786-085 OneQuant™ HOOK™ Iodoacetyl-LC-Biotin 8 x 2mg Optimizer Bu!er™ ™ ™ Protein 786-698 OneQuant HOOK Sulfo-NHS-Biotin 8 x 1mg For enhanced coupling e!ciency 786-699 OneQuant™ HOOK™ Sulfo-NHS-SS-Biotin 8 x 1mg ™ ™ 786-700 OneQuant HOOK NHS-dPEG4-Biotin 8 x 1mg HOOK™ Biotin Kits """"""""""""""""" SpinOUT™ Column For highly e#cient labeling of proteins Rapid (<10min) Puri"cation HOOK™ Biotin kits come with all the necessary reagents, equipment and instructions for optimization of reaction conditions, e"cient labeling, removal of unbound biotin and quanti!cation of biotin Centrifuge to recover labeled protien labeling. In addition to highly e"cient labeling, the HOOK™ Biotin kits o$er the advantage of being supplied with SpinOUT™ desalting 0

™ 0 )/ HABA-Avidin Estimate Biotin columns and a speci!c Optimizer Bu$er . These simplify the labeling /) / Assay Labeling E!ciency ) process and ensure high levels of biotin labeling. 4 PROTEIN LABELING Figure 24: HOOK™ Biotin kit scheme. Each kit is supplied with 25mg of speci!c HOOK™ Biotin Reagent that conjugates to proteins through amines, sulfhydryls, carboxyls FEATURES or carbohydrates. The amine and sulfhydryl coupling HOOK™ Biotin t 0QUJNJ[FS#VòFS™ for improved coupling e"ciency Reagents couple directly to the protein through their reactive groups, t 4QJO065™ gel !ltration columns for rapid (<10 minute) puri!cation however the carboxyl coupling HOOK™ Biotin Reagents require a t #JPUJOBTTBZSFBHFOUTUPEFUFSNJOFMFWFMPGCJPUJOJODPSQPSBUJPO carbodiimide crosslinker and the carbohydrate coupling HOOK™ Biotin t -BCFMTNHQSPUFJOSFBDUJPO Reagents require carbohydrate oxidation before coupling. The HOOK™ t 4VJUBCMFGPSDPVQMJOHSFBDUJPOT Biotin kits include EDC as the carbodiimide crosslinker in the carboxyl coupling kits and sodium meta-periodate for carbohydrate oxidation $BU Description Size in the carbohydrate coupling kits. BS-01 HOOK™-NHS-Biotin Kit 10 reactions In addition to the above, each HOOK™ Biotin kit contains a speci!c BS-02 HOOK™-NHS-LC-Biotin Kit 10 reactions Optimizer Bu$er that provides the optimal reaction conditions for BS-03 HOOK™-NHS-LC-LC-Biotin Kit 10 reactions each HOOK™ Biotin Reagent. BS-04 HOOK™-NHS-SS-Biotin Kit 10 reactions ™ ™ PURIFICATION BS-05 HOOK -NHS-dPEG4 -Biotin Kit 10 reactions Following the labeling of the protein with the HOOK™ Biotin BS-06 HOOK™-sulfo-NHS-Biotin Kit 10 reactions Reagent the unreacted biotin and other chemicals are rapidly BS-07 HOOK™-sulfo-NHS-LC-Biotin Kit 10 reactions ™ removed from the labeled protein with the supplied SpinOUT BS-08 HOOK™-sulfo-NHS-LC-LC-Biotin Kit 10 reactions columns. These columns use gel !ltration to remove the by-products BS-09 HOOK™-sulfo-NHS-SS-Biotin Kit 10 reactions in <10 minutes. BS-10 HOOK™-PFP-Biotin Kit 10 reactions BIOTIN ESTIMATION ™ BS-11 HOOK -PEG2-Iodoacetyl-Biotin Kit 10 reactions ™ HOOK BiotinQuant measures biotin using HABA BS-12 HOOK™-Iodoacetyl-LC-Biotin Kit 10 reactions [4’-hydroxyazobenzene-2-carboxylic acid] dye. HABA binds with BS-13 HOOK™-Biotin-PDA Kit 10 reactions avidin at the biotin-binding site. A characteristic color, that absorbs BS-14 HOOK™-Biotin-BMCC Kit 10 reactions at 500nm, is produced (ε=35,500 M-1cm-1 expressed as per mole of ™ HABA bound). Biotin or biotinylated agents compete with the HABA BS-15 HOOK -Biotin-Pentylamine Kit 10 reactions BS-16 HOOK™-Biotin-PEG -Amine Kit 10 reactions for the binding sites and the greater a"nity biotin reagents displace 2 ™ HABA from the avidin binding sites and proportionally reduce the BS-17 HOOK -Biotin-PEG3-LC-Amine Kit 10 reactions absorbance. The HOOK™ BiotinQuant kit is supplied with each HOOK™ BS-18 HOOK™-Biotin-Hydrazide Kit 10 reactions Biotin Kit and is also available separately. The HABA dye is also BS-19 HOOK™-Biotin-LC-Hydrazide Kit 10 reactions available separately.

For further details, visit www.GBiosciences.com 19 Antibody Labeling Micro HOOK™ Biotin Kits HOOK™ IgG Biotinylation """"""""""""""""" """"""""""""""""" For highly e#cient labeling of proteins Rapid antibody labeling with biotin The micro HOOK™ Biotin kits are designed to label small amounts Designed for the e"cient biotinylation of IgG molecules by !rst of proteins, with each kit designed for 8-10 labelings of 50-250µg immobilizing the IgG molecules on a solid support protein/reaction. Each kit is supplied with all the necessary reagents The HOOK™ IgG Biotinylation kits o$er an advantage over standard for optimization of reaction conditions, e"cient labeling and removal biotinylation reactions as the immobilization of the IgG to the Nickel of unbound biotin. In addition to highly e"cient labeling, the HOOK™ Chelating resin allows for the rapid removal of uncoupled biotin and Biotin kits o$er the advantage of being supplied with SpinOUT™ therefore eliminates the need for further dialysis or desalting of the desalting columns and a speci!c Optimizer Bu$er™. These simplify the biotinylated antibody. labeling process and ensure high levels of biotin labeling. Two kits are available for labeling antibodies through free amines

PROTEIN LABELING or sulfhydryls. The amine kit uses NHS-dPEG4-Biotin to label free Each kit is supplied with 8 x 1mg single use aliquots of biotin primary amines. The sulfhydryl kit uses the supplied Protein-S-S- ™ reagent to minimize waste and degradation of the NHS ester coupling Reductant to reduce the disul!de bonds of the immobilized IgG reaction group. The following HOOK™ Biotin reagents are available in molecule. The reduced immobilized IgG molecule is then incubated with PEG -Iodoacetyl-Biotin solution to biotinylate the free sulfhydryl the micro format: 2 groups. ™ t HOOK Sulfo-NHS-Biotin The advantage of a PEG (polyethylene glycol) biotinylation reagent Amine reactive reagent, shortest spacer arm is that the long hydrophilic spacer arm conveys its water solubility ™ t HOOK Sulfo-NHS-LC-Biotin to the antibodies and have a reduced occurrence of aggregation Amine reactive reagent, longer spacer arm compared to non-PEG biotinylation reactions. t HOOK™ Sulfo-NHS-SS-Biotin

H 2 -S-S- 2 N- -NH Cleavable, amine reactive reagent -S-S- N- -NH H 2 t HOOK™ NHS-dPEG -Biotin 2 4 Immobilize antibody on -NH Amine reactive, pegylated reagent; enhances water solubility 2 Nickel Column Histidine

H N- O 2 Rich Region NH ™ HN O H O O N O In addition, each HOOK Biotin kit contains a speci!c Optimizer O S -S-S- O N O O O H N- -NH2 ™ 2 + Bu$er that provides the optimal reaction conditions. -S-S- PURIFICATION

™ S

H N

O O Following the labeling of the protein with the HOOK Biotin O

O O H -S-S- 2 N-

O O

O O -N

O HN

O N -S-S-

H -N H N- N O O

Reagent the unreacted biotin and other chemicals are rapidly HN O O S

O O ™ O

removed from the labeled protein with the supplied SpinOUT O

NH HN

H O O N O O S

-N O Columns. These columns use gel !ltration to remove the by-products O Add Biotin Reagent, H N- Wash Away Unbound Biotin 2

in <10 minutes. O

-S-S- NH HN

H O O N O O S

O -N O

N- O O -S-S-

FEATURES O

N H

t .JDSPLJUGPSMBCFMJOHQSPUFJOQSJNBSZBNJOFT O

S O t 0QUJNJ[FS#VòFS™ for improved coupling e"ciency NH

H t (FMöMUSBUJPODPMVNOTGPSSBQJE NJOVUF QVSJöDBUJPO N O O O

O O H -S-S- 2 N-

O O

O O -N

NH O

O HN

O N -S-S-

t -BCFMTHQSPUFJOSFBDUJPO H -N H N- N O O

HN O O S NH

O O t 4VJUBCMFGPSDPVQMJOHT Elute O

NH HN

H O O N O O S

-N O O Biotinylated Antibody

H N- $BU Description Size 2

-S-S- NH ™ HN

H O O N O O S

786-694 HOOK -sulfo-NHS-Biotin Kit (micro) 8-10 reactions O -N O

N- O O -S-S-

™ O

786-695 HOOK -sulfo-NHS-LC-Biotin Kit (micro) 8-10 reactions O

N H

™ O HN

786-696 HOOK Sulfo-NHS-SS-Biotin Kit (micro) 8-10 reactions S

O NH ™ ™ 786-697 HOOK -NHS-dPEG4-Biotin Kit (micro) 8-10 reactions Figure 25: HOOK IgG Biotinylation (Amine) Scheme. The IgG antibody is "rst immobilized through its histidine rich domain on a nickle column. ™ Immobilized antibody is labeled with the NHS-dPEG -Biotin reagent that HOOK BiotinQuant 4 """"""""""""""""" reacts with primary amines. Free biotin is washed away and the biotinylated For the estimation of biotin conjugation antibody is eluted with the supplied His Elution Bu#er. HOOK™ BiotinQuant measures biotin using HABA FEATURES [4’-hydroxyazobenzene-2-carboxylic acid] dye. HABA binds with t 4JNQMFSBOUJCPEZCJPUJOZMBUJPO avidin at the biotin-binding site. A characteristic color, that absorbs t 4PMJETVQQPSUUFDIOPMPHZFMJNJOBUFTEJBMZTJTEFTBMUJOH at 500nm, is produced (ε=35,500 M-1 cm-1 expressed as per mole of t 4VJUBCMFGPSNHBOUJCPEZ HABA bound). Biotin or biotinylated agents compete with the HABA t 1&(#JPUJOSFBHFOUGPSSFEVDFETUFSJDIJOESBODFBOEJODSFBTFE for the binding sites and the greater a"nity biotin reagents displace labeled antibody solubility HABA from the avidin binding sites and proportionally reduce the APPLICATIONS absorbance. t 'PSUIFFóDJFOUBOETJNQMFMBCFMJOHPGBOUJCPEJFTXJUICJPUJO

$BU Description Size $BU Description Size BKC-01 HOOK™ BiotinQuant Kit 20 assays 786-728 HOOK™ IgG Biotinylation (Amine) 8 reactions BKC-03 HABA Dye 1g 786-729 HOOK™ IgG Biotinylation (Sulfhydryl) 8 reactions

20 For further details, visit www.GBiosciences.com Antibody Labeling BIOTIN CONJUGATION ESTIMATION BIOTIN PURIFICATION """"""""""""""""" """"""""""""""""" HOOK™ BiotinQuant Streptavidin Resin """"""""""""""""" """"""""""""""""" For the estimation of biotin conjugation High binding a#nity for biotin labeled proteins & HOOK™ BiotinQuant measures biotin using HABA molecules [4’-hydroxyazobenzene-2-carboxylic acid] dye. HABA binds with #JPUJO B%BWJUBNJO 7JUBNJO) NPMFDVMF FYIJCJUTBO avidin at the biotin-binding site. A characteristic color, that absorbs extraordinary binding a"nity for avidin (Ka=1015 M-1) and streptavidin -1 -1 at 500nm, is produced (ε=35,500 M cm expressed as per mole of (Ka=1015 M-1). Biotin and (strept)avidin interaction is rapid and HABA bound). Biotin or biotinylated agents compete with the HABA once the bond is established it can survive up to 3M guanidine- for the binding sites and the greater a"nity biotin reagents displace hydrochloride and extremes of pH. Biotin-avidin bonds can only HABA from the avidin binding sites and proportionally reduce the be reversed by denaturing the avidin protein molecule with 8M absorbance. guanidine-hydrochloride at pH1.5 or by boiling in SDS Page Sample $BU Description Size Loading Bu$er. Streptavidin is a tetrameric protein containing 4 biotin binding BKC-01 HOOK™ BiotinQuant Kit 20 assays sites. Streptavidin in many respects is similar to avidin except that BKC-03 HABA Dye 1g it has no carbohydrate and has a slightly lower molecular weight of about 60kDa. The solubility of streptavidin (isoelectric pH5) Avidin in aqueous bu$er is much lower than avidin, but the binding of """"""""""""""""" streptavidin to biotin is similar to that of avidin. The advantage of A#nity puri"ed for the estimation of biotin streptavidin is that the lack of carbohydrates signi!cantly reduces the conjugation amount of non-speci!c binding. 5IFTUSFQUBWJEJOVTFEGPSJNNPCJMJ[BUJPOPOQPSPVTDSPTTMJOLFE "WJEJOJTBHMZDPQSPUFJOXJUIBQQSPYJNBUFMZPGJUTUPUBMNBTT agarose is a recombinant form with a mass of 53kDa and near neutral comes from carbohydrates. Avidin has a molecular weight of 67kDa pI. The streptavidin in covalently coupled to the agarose resulting in and contains four identical 128 amino acid subunits that each have minimal leaching and is stable over pH2-11. a single biotin binding domain. Avidin is a basic protein with an The Steptavidin Resin is designed for the single step small and isoelectric pH of 10-10.5 and is readily soluble in aqueous bu$ers large scale a"nity puri!cation of proteins and antibodies with a biotin containing a wide range of salt, pH, temperature and other laboratory tag. The resin can also be used for immunoprecipitations using biotin agents. This wide range of tolerance makes avidin suitable for a wide labeled antibodies. Supplied as a resin slurry or in a 1ml spin column variety of analytical applications. format. This a"nity puri!ed avidin is ideal for estimation of biotin Speci!c Binding and Elution Bu$ers are also available. incorporation and other applications. The Streptavidin Resin is available as resin alone or supplied in a kit $BU Description Size format containing: BKC-02 Avidin 2 x 5mg t NMSFTJO t NM4USFQUBWJEJO#JOEJOH8BTI#VòFS

(20mM NaPO4, 0.15M NaCl, pH7.5) HABA t NM4USFQUBWJEJO&MVUJPO#VòFS .(VBOJEJOF)$MQ) """"""""""""""""" t FNQUZNMTQJODPMVNOT A biotin estimation dye reagent. t FNQUZNMHSBWJUZøPXDPMVNOT $BU Description Size The bu$ers are also available separately. BKC-03 HABA 1g FEATURES t 3FDPNCJOBOUTUSFQUBWJEJODPWBMFOUMZDPVQMFEUP_DSPTTMJOLFE agarose. Minimal Leaching t -JHBOE%FOTJUZ NHNM t #JOEJOHDBQBDJUZHCJPUJONMSFTJO APPLICATIONS t *NNVOPQSFDJQJUBUJPOXJUICJPUJOZMBUFEBOUJCPEJFT t 1VMMEPXOBTTBZTXJUICJPUJOZMBUFEQSPUFJOT t 1VSJöDBUJPOPGCJPUJOZMBUFENPMFDVMFT JODMVEJOH t 1SPUFJOT t "OUJCPEJFT t %/" t $BSCPIZESBUFT

$BU Description Size 786-590 Immobilized Streptavidin Resin 2ml resin 786-390 Immobilized Streptavidin Resin 5ml Resin 786-591 Immobilized Streptavidin Resin 10ml resin 786-592 Immobilized Streptavidin Resin 5 x 1ml 786-555 Streptavidin Resin Kit 1 786-548 Streptavidin Binding Bu$er 100ml 786-549 Streptavidin Elution Bu$er 100ml For further details, visit www.GBiosciences.com 21 Antibody Labeling Avidin Resin Monomeric Avidin Resin """"""""""""""""" """"""""""""""""" High binding a#nity for biotin labeled proteins & Puri"cation & elution of biotin labeled molecules molecules under mild elution conditions #JPUJO B%BWJUBNJO 7JUBNJO) NPMFDVMF FYIJCJUTBO G-Biosciences Immobilized Monomeric Avidin Resin is designed extraordinary binding a"nity for avidin (Ka=1015 M-1). Biotin and for the simple a"nity chromatography puri!cations of proteins, avidin interaction is rapid and once the bond is established it can antibodies and other molecules with a biotin tag. The resin consists survive up to 3M guanidine-hydrochloride and extremes of pH. Biotin- PGNPOPNFSJDTVCVOJUTPGBWJEJODPWBMFOUMZDPVQMFEUPDSPTT avidin bonds can only be reversed by denaturing the avidin protein linked agarose, o$ering a stable, reusable resin for the puri!cation of molecule with 8M guanidine-hydrochloride at pH1.5 or by boiling in biotinylated molecules. SDS Page Sample Loading Bu$er. Monomeric avidin o$ers a distinct advantage over native avidin, "WJEJOJTBHMZDPQSPUFJOXJUIBQQSPYJNBUFMZPGJUTUPUBMNBTT a tetrameric molecule, and streptavidin as it has a much lower biotin coming from carbohydrates. Avidin has a molecular weight of 67kDa binding a"nity, Kd=10-7 as opposed to Kd=10-15 for native avidin. This and contains four identical 128 amino acid subunits that each has lower binding a"nity allows elution of molecules with mild elution a single biotin binding domain. Avidin is a basic protein with an bu$ers (2mM D-Biotin in 1X PBS), as opposed to the strong denaturing isoelectric pH of 10-10.5 and is readily soluble in aqueous bu$ers CVòFST .(VBOJEJOFt)$M Q) VTFEXJUIOBUJWFBWJEJO containing a wide range of salt, pH (2-11), temperature and other The covalent attachment of monomeric avidin to the agarose laboratory agents. This wide range of tolerance makes avidin suitable ensures no detectable leaching of the avidin during biotin puri!cation for a wide variety of analytical applications. Avidin has extraordinary and o$ers a wide tolerance to chemicals. This ensures the resin can be binding a"nity for biotin (Ka=1015M-1). reused at least 10 times with no loss of function. The avidin in covalently coupled to the agarose resulting in 5IF*NNPCJMJ[FE.POPNFSJD"WJEJO3FTJOJTBWBJMBCMFBTB minimal leaching and is stable over pH2-11. resin slurry or as a complete kit containing a reusable monomeric The Avidin Resin is designed for the single step small and large avidin column and the respective bu$ers for successful puri!cation of scale a"nity puri!cation of proteins and antibodies with a biotin tag. biotinylated molecules. The resin can also be used for immunoprecipitations using biotin FEATURES MBCFMFEBOUJCPEJFT4VQQMJFEBTBSFTJOTMVSSZ t .POPNFSJDBWJEJODPWBMFOUMZDPVQMFEUP_DSPTTMJOLFEBHBSPTF Speci!c Binding and Elution Bu$ers are also available. t .JOJNBM-FBDIJOH FEATURES t #JOEJOHDBQBDJUZxNHCJPUJOZMBUFE#4"NMSFTJO t "WJEJODPWBMFOUMZDPVQMFEUP_DSPTTMJOLFEBHBSPTF.JOJNBM t /PO%FOBUVSJOH&MVUFCJPUJOZMBUFENPMFDVMFTXJUIGSFFCJPUJO Leaching t 3FVTBCMF3FVTFUIFSFTJOBUMFBTUUJNFT MPTTPGCJOEJOH t #JOEJOHDBQBDJUZHCJPUJONMSFTJO regeneration) t 4QFDJöD3FUBJOTBWJEJOTIJHITQFDJöDJUZGPSCJPUJONPMFDVMFT APPLICATIONS t *NNVOPQSFDJQJUBUJPOXJUICJPUJOZMBUFEBOUJCPEJFT APPLICATIONS t 1VMMEPXOBTTBZTXJUICJPUJOZMBUFEQSPUFJOT t 1VSJöDBUJPOPGCJPUJOZMBUFENPMFDVMFT JODMVEJOH t 1VSJöDBUJPOPGCJPUJOZMBUFENPMFDVMFT JODMVEJOH t 1SPUFJOT t 1SPUFJOT t "OUJCPEJFT t "OUJCPEJFT t %/" t %/" t $BSCPIZESBUFT t $BSCPIZESBUFT $BU Description Size $BU Description Size 786-595 Immobilized Monomeric Avidin 5ml resin 786-593 Immobilized Avidin Resin 5ml resin 786-596 Immobilized Monomeric Avidin 10ml resin 786-594 Immobilized Avidin Resin 25ml Resin 786-597 Immobilized Monomeric Avidin Kit 786-548 Streptavidin Binding Bu$er 100ml 786-549 Streptavidin Elution Bu$er 100ml

22 For further details, visit www.GBiosciences.com Antibody Puri!cation Accessories ENZYME LABELING DISPOSABLE COLUMNS """"""""""""""""" """""""""""""""""

HOOK™ HRP PLUS Labeling Spin Column, <0.1ml """"""""""""""""" """"""""""""""""" The HOOK™ HRP PLUS labeling kit is a high e"ciency enzyme labeling kit for tagging proteins with horseradish peroxidase enzyme. This LJUIBTBOBDUJWBUFE)31UIBUDPVQMFTXJUIIJHIFóDJFODZ UP the numerous amine groups of proteins and is superior to the commonly used glutaraldehyde coupling chemistry. This kit uses HOOK™ HRP PLUS, which is HRP that has been activated by the addition of reactive aldehydes. The aldehyde groups react spontaneously and at high e"ciency with primary amines, located at the N-terminus of proteins or in lysine residues, to form intermediate Schi$ Base complexes. These, in turn, are selectively reduced by Figure 26: Spin Column, <0.1ml. the supplied reduction agent. Following quenching of the reaction Unique design with the snap o$ end converting to a closure for the the protein is linked to the horseradish peroxidase enzyme by stable column for easy manipulation and use. amine linkage. The labeled protein, or antibody, can now be used for FEATURES immunoblotting, ELISA and histochemical techniques. t $PMVNOWPMVNFM FEATURES t 3FTJOWPMVNFM t "DUJWJUZJTVOJUTNH t 'JMUFSUZQF1PMZFUIZMFOFöMUFS _NQPSFTJ[F t 3FBDUTXJUIQSJNBSZBNJOFTUPGPSNDPWBMFOUBNJOFCPOET t 'JUTJOBOENMDFOUSJGVHFUVCFT t 4UBCMFGPS NPOUITBU¡$ Cat Description Size $BU Description Size 786-718 Spin Column, <0.1ml 25 ™ 786-313 HOOK HRP PLUS labeling kit 5 reactions 786-719 Spin Column, <0.1ml 50 HOOK™ HRP Sulfo Labeling Spin Column, 1ml """"""""""""""""" """"""""""""""""" An e"cient enzyme labeling kit for tagging proteins with horseradish peroxidase (HRP) enzyme. This kit has activated HRP that couples to peptides, proteins and ligands that have free sulfhydryl groups. The maleimide activated HRP saves time as the !rst step of the normal two-step maleimide activation procedure is already complete, saving several hours of valuable research time. To aid in the preparation of HRP conjugates using free sulfhydryls the kit is supplied with SATA (N-Succinimidyl S-acetylthioacetate), to add free sulfhydryls to existing amine groups, and 2-mercaptoethylamine.HCl, a mild reducing agent for conjugating HRP to immunoglobulin G (IgG) and its fragments. Figure 27: Spin Column, 1ml. FEATURES FEATURES t 3FBDUTXJUIGSFFTVMGIZESZMTUPGPSNDPWBMFOUCPOET t $PMVNOWPMVNFNM t 4VQQMJFEXJUISFBHFOUTUPHFOFSBUFTVMGIZESZMHSPVQT t 3FTJOWPMVNFM t 'JMUFSUZQF1PMZFUIZMFOFöMUFS _NQPSFTJ[F $BU Description Size t $BQBOESVCCFSTUPQQFSTJODMVEFE 786-314 HOOK™ HRP SULFO labeling kit 5 reactions t 'JUTJOBOENMDFOUSJGVHFUVCFT HOOK™ AP Sulfo Labeling Cat Description Size """"""""""""""""" 786-198 Spin Column, 1ml 10 An e"cient enzyme labeling kit for tagging proteins with alka- 786-720 Spin Column, 1ml 25 line phosphatase enzyme. This kit has activated AP that couples to 786-721 Spin Column, 1ml 50 peptides, proteins and ligands that have free sulfhydryl groups. The maleimide activated AP saves time as the !rst step of the normal two-step maleimide activation procedure is already complete, saving several hours of valuable research time. To aid in the preparation of AP conjugates using free sulfhydryls the kit is supplied with SATA, to add free sulfhydryls to existing amine groups, and 2-mercaptoethylamine.HCl, a mild reducing agent for conjugating AP to immunoglobulin G (IgG) and its fragments. FEATURES t 3FBDUTXJUIGSFFTVMGIZESZMTUPGPSNDPWBMFOUCPOET t 4VQQMJFEXJUISFBHFOUTUPHFOFSBUFTVMGIZESZMHSPVQT

$BU Description Size 786-315 HOOK™ AP SULFO labeling kit 5 reactions For further details, visit www.GBiosciences.com 23 Spin Column, 2ml Spin Column, 5ml """"""""""""""""" """""""""""""""""

Figure 28: Spin Column, 2ml. FEATURES t $PMVNOWPMVNFNM t 3FTJOWPMVNFNM Figure 30: Spin Column, 5ml. t 'JMUFSUZQF1PMZFUIZMFOFöMUFS _NQPSFTJ[F t $BQBOESVCCFSTUPQQFSTJODMVEFE FEATURES t 'JUTNMDPOJDBMDFOUSJGVHFUVCFT t 5PUBMWPMVNFNM t 3FTJOWPMVNFNM Cat Description Size t 'JMUFSUZQF QPSFTJ[F1PMZFUIZMFOFöMUFS _NQPSFTJ[F 786-722 Spin Column, 2ml 25 t 'JUTNMDPOJDBMDFOUSJGVHFUVCFT 786-723 Spin Column, 2ml 50 t $BQBOESVCCFSTUPQQFSTJODMVEFE Spin Column, 3ml Cat Description Size """"""""""""""""" 786-726 Spin Column, 5ml 10 Spin Column, 10ml 21.5mm """""""""""""""""

2ml 20mm reservoir

79mm 3ml 46.5mm resin bed volume

12.5mm

10.5mm Figure 29: Spin Column, 3ml.

FEATURES t $PMVNOWPMVNFNM t 3FTJOWPMVNFNM t 'JMUFSUZQF1PMZFUIZMFOFöMUFS _NQPSFTJ[F t $BQBOESVCCFSTUPQQFSTJODMVEFE t 'JUTNMDPOJDBMDFOUSJGVHFUVCFT

Cat Description Size 786-724 Spin Column, 3ml 25 786-725 Spin Column, 3ml 50 Figure 31: Spin Column, 10ml. FEATURES t 5PUBMWPMVNFNM t 3FTJOWPMVNFNM t 'JMUFSUZQF QPSFTJ[F1PMZFUIZMFOFöMUFS _NQPSFTJ[F t 'JUTNMDPOJDBMDFOUSJGVHFUVCFT t $BQBOESVCCFSTUPQQFSTJODMVEFE

Cat Description Size 786-727 Spin Column, 10ml 10 24 For further details, visit www.GBiosciences.com G-Biosciences Product Line Overview

c www.GBiosciences.com Updated: Jul 28, 2011 XXX(#JPTDJFODFTDPN