Epitope Tags in Protein Research Tag Selection & Immunotechniques Biomapping

biomapping

Epitope Tags in Protein Research Tag Selection & Immunotechniques biomapping

Contents Choosing an Epitope Tag ...... 3 Immunoprecipitation ...... 31 Purification ...... 3 Reagents ...... 32 Detection ...... 5 Direct Immunoprecipitation (microcolumn) ...... 33 Cleavage sites ...... 8 Large scale (5 mL) procedure ...... 34 High Throughput Expression ...... 8 Indirect Immunoprecipitation ...... 35 Summary ...... 9 ‘Resin First’ method ...... 36 References ...... 9 ‘Lysate First’ method ...... 37 Epitope Tags ...... 10 Preparation for SDS-PAGE ...... 38 Cellulose Binding Domain (CBD) ...... 10 Immunoblotting (Western Blotting) ...... 38 Chloramphenicol Acetyl Transferase (CAT) ...... 10 Reagents ...... 39 Dihydrofolate Reductase (DHFR) ...... 11 Protein Transfer ...... 39 FLAG® ...... 11 Direct Detection ...... 40 Glutathione S-Transferase (GST) ...... 13 Indirect Detection ...... 41 Green Fluorescent Protein (GFP) ...... 14 Immunohistochemistry ...... 42 Hemagglutinin A (HA) ...... 14 Reagents and Equipment ...... 42 Histidine (His) ...... 15 Paraffin Removal/Rehydration ...... 43 Herpes Simplex Virus (HSV) ...... 16 Antigen Retrieval (optional step for weak signal) . . . 44 Luciferase ...... 16 Enzymatic Method ...... 44 Maltose-Binding Protein (MBP) ...... 17 Microwave Retrieval ...... 44 c-Myc ...... 17 Inactivation of Peroxidase (if HRP detection used) . .45 Protein A and Protein G ...... 18 Primary Antibody Reaction ...... 46 Streptavidin ...... 18 Secondary Reaction ...... 47 T7 ...... 20 Option 1 Biotin/Streptavidin Detection ...... 47 Thioredoxin ...... 21 Option 2 Enzyme-labeled Secondary Antibody . . . 48 V5 ...... 21 Development ...... 49 Vesicular Stomatitis Virus Glycoprotein (VSV-G) . . . . 22 Counterstaining ...... 50 Yeast 2-hybrid tags: B42, GAL4, LexA, VP16 ...... 22 Immunofluorescence ...... 51 Further reading ...... 23 Reagents and Equipment ...... 52 Protocols ...... 24 Cell Preparation ...... 53 Protein Extraction ...... 24 Fixation ...... 53 Sonication (E . coli) ...... 24 Methanol-Acetone Fixation ...... 53 Reagents ...... 24 Paraformaldehyde-Triton® Fixation ...... 53 Procedure ...... 25 Paraformaldehyde-Methanol Fixation ...... 54 Freeze-thawing (cultured cells) ...... 26 PEM-Ethanol Fixation ...... 54 Reagents ...... 26 Application of Primary Antibody ...... 55 Procedure ...... 26 Application of Secondary Antibody ...... 55 Detergent extraction (S . cerevisiae) ...... 27 Evaluation ...... 56 Reagents ...... 27 ELISA ...... 56 Procedure ...... 27 Indirect ELISA ...... 56 Protein Purification ...... 28 Reagents and Equipment ...... 56 His-tagged protein purification ...... 28 Antigen Coating ...... 57 Reagents ...... 28 Primary Antibody Reaction ...... 58 Procedure ...... 29 Development ...... 59 Reagent compatibility chart ...... 30 References ...... 59 Order 800-325-3010 Technical Service 800-325-5832 3

Choosing an Epitope Tag Unlike DNA and RNA, which can be targeted with complementary oligonucleotides, protein detection and purification usually relies upon specific antibodies . Although many antibodies are available commercially, they do not cover all proteins, especially if the protein has novel or unknown sequences . Raising polyclonal antibodies is time-consuming and expensive; raising monoclonal antibodies even more so . Epitope tags (also known as fusion tags or affinity tags) offer a convenient solution to this problem by acting as universal epitopes for detection and purification without disturbing the structure of the protein to which they are fused . One of the earliest tags was poly-arginine; a run of five arginine residues created a basic region that allowed purification with cation exchange resin (Smith et al ., 1984) . Since then, the number of tags has steadily grown, and so have the number of factors that can be considered when designing an expression study . This guide lays out the features of different epitope tags to help with this design; one of the main choices is whether the aim of expression is detection or purification of the protein .

Purification All tags offer some means of purification, but the purity, convenience, and cost of these platforms determine their suitability . The best known tag is poly-histidine, which is a form of Immobilized Metal Affinity Chromatography (IMAC) . It is based upon the affinity of nickel for four or more consecutive histidine residues . It is popular because of its ability to purify under denaturing conditions, ease of reuse, and reasonable price .

-OOC -OOC N Ni C Resin N: :N H N: CH 2 N -OOC Coordinated Bond

2 Protein CH Nickel(II) ion

Figure 1: Binding of Histidine repeats to immobilized Nickel ion via coordinated bonds . biomapping

Other tags with cost-effective purification resins are Maltose-Binding Protein (MBP), Cellulose-Binding Domain (CBD), and Glutathione S-Transferase (GST) . These tags are approximately 40 times the size of metal affinity tags, so they may interfere with the structure or function of the fusion partner . However, this can be beneficial: tags such as MBP and GST can improve solubility (Dyson et al ., 2004), which is especially important when expressing proteins at high levels in prokaryotes as they have more primitive post- translational folding and processing machinery than eukaryotes . Tags that use antibody-based purification formats (e .g ., FLAG®, HA, HSV, c-Myc) offer higher levels of purity, but at a cost: antibody resins are expensive to produce and cannot be reused easily . However, the high-purity often means that they are cost-effective in the long run as secondary purification steps can be avoided . This is of particular use in high-throughput screening, or where proteins are expressed at low levels, e .g ., while expression conditions are being optimized . Epitope tags can also be used for immobilization or attachment, e .g ., to investigate protein interactions . If a purification resin is loaded with protein, then it can be used as ‘bait’ to capture binding partners (immunoprecipitation) . In this case, the specificity of the resin becomes crucial to avoid interference from contaminants .

-14 Biotin-based tags bind very tightly (Ka = 10 M) to streptavidin (Bayer et al ., 1990) so they are ideal for immobilizing proteins for studies where they will be subjected to repeated washes (e .g ., ELISA and array- based assays) .

Detection The ideal tag for detection would be large and hydrophilic, with a strong antibody recognition site posi- tioned in an exposed region of the protein . However, large tags can interfere with protein structure, and exposed regions are often functionally active, so choosing a tag is not trivial . Because detection is performed under aqueous conditions, a hydrophilic tag is more likely to be presented at the protein surface and thus, accessible to antibodies and capture agents . All tags are hydrophilic but some more than others . Possession of charged (acid or basic) residues increases hydrophilicity and is a feature of naturally-ocurring epitopes . The hydrophilic nature of tags can also increase the solubility of the expression protein; size and posi- tioning of the tag also contributes to this effect (Dyson et al ., 2004) . Large tags are often easier to detect because they are sterically more accessible to antibodies . Even in the presence of SDS, smaller tags can sometimes be folded inside the host protein and evade detection . Using a tag that incorporates charged residues helps overcome this; their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition . The FLAG® epitope is a practical example of how size and charge phenomena can be exploited; it is only eight amino acids long but all residues are charged (DYKDDDDK), so detection is very sensitive . This sensitivity is enhanced 10-fold by tripling the size of the epitope to the ‘3xFLAG™’ tandem repeat (DYKDHDGDYKDHDIDYKDDDK) . Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein, but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis . However, N-terminal placement seems to have a stronger effect on protein solubility, possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al ., 2004) . Detection need not be restricted to immunodetection . Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays, respectively . GFP can also be imaged in live cells, so processes can be studied in real time rather than extrapolated from fixed sections . A comparison of different detection methods is shown in the figures below .

Comparison of Immunodetection Methods Immunoblotting Immunofluorescence

Substrate Signal viewed by Horseradish Fluorophore conjugated fluoresence to secondary microscopy peroxidase (HRP) antibody antibody conjugate Oxidized substrate + signal (light or color change) Primary antibody

EpitopeEpitope tag PrProteinotein Protein Epitope tag

Tissue section fixed onto glass slide Blotting membrane

Immunocytochemistry ELISA (Enzyme-Linked ImmunoSorbent Assay)

Substrate Substrate

Enzyme conjugated Colored Enzyme conjugated Colored to secondary precipitate to secondary precipitate antibody antibody

Primary antibody Primary antibody

Protein in solution (e.g., cell lysate) EpEpitopeitope tag Proteoteinin Epitope tag

Reaction vessel (e.g., 96-well plate) Tissue section fixed onto glass slide biomapping

Cleavage Sites Although small tags may not interfere with folding, it is often better to work with the native proteins, especially if attempting to determine the structure . To this end, there are a number of proteases and site combinations available for removal of tags . These sites are often pre-engineered into expression vectors, and in the case of enterokinase, actually form part of the tag sequence . Proteases can be heat-inactivated post-cleavage, or more commonly used as resin conjugates for efficient removal .

Cleavage Site Enzyme Asp-Asp-Asp-Asp-Lys-X Enterokinase Ile-Glu/Asp-Gly-Arg-X Factor Xa Leu-Val-Pro-Arg-X-Gly-Ser Thrombin Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal .

High-Throughput Expression Because protein expression is still an inexact science, even small expression projects can require hundreds of combinations given all of the factors that can be assessed . Choice of epitope tag can influence how easy these experiments are to carry out both practically (e .g ., are multiwell purification plate formats available for screening?) and scientifically (e .g ., will it be possible to detect the protein at the expected levels of expression?) . To this end, it can be useful to incorporate more than one tag, typically at opposite termini to avoid the risk of interference . An example of this might be to use a sensitive ‘detection’ tag as well as a ‘purification’ tag so that expression can be optimized quickly using the detection tag, yet purified with the purification tag without the need to reclone . For large expression projects it becomes impractical to test many combinations for each protein, and this has led to studies to predict optimal parameters for successful expression . One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility . Many eukaryotic proteins require post-translational modifications in order to fold correctly, which coupled with the artificially high protein concentrations in the cell can lead to misfolding and aggregation . Accumulation of misfolded protein is often toxic, but even if the proteins are packaged in inclusion bodies, refolding them is not trivial . Order 800-325-3010 Technical Service 800-325-5832 7

Summary Epitope tags simplify protein expression studies by providing universal methods for purification and detection . They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation . Choice of tag depends upon many factors, and involves a degree of compromise in most cases, but one of the most important considerations is the downstream analysis of the protein . ‘Purification’ tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection . ‘Detection’ tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification . One compromise solution is combinatorial tagging; using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies .

References Bayer, EA; Ben-Hur, H .; Wilchek, M . (1990) “Isolation and properties of streptavidin ”. Methods Enzymol., 184: 80–9 . Dyson, MR; Shadbolt, SP; Vincent, KJ; Perera, RL; McCafferty, J . (2004) “Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression ”. BMC Biotechnol., Dec 14; 4(1), 32 . Hernan, R .; Heuermann, K .; Brizzard, B . (2000) “Multiple epitope tagging of expressed proteins for enhanced detection ”. Biotechniques, Apr, 8(4), 789–93 . Smith, JC; Derbyshire, RB; Cook, E; Dunthorne, L; Viney, J; Brewer, SJ; Sassenfeld, HM; Bell LD . (1984) “Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification ”. Gene, Dec., 32(3), 321–7 . biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized, along with the tools available for their study .

Legend Detection Purification Controls

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene, and retains its functionality when fused to most proteins . This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection . Purification is achieved using chloramphenicol-Sepharose® .

Cat . No . Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit C9336- 5ML.

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway . Purification of tagged proteins can be achieved by methotrexate-linked resin .

Cat . No . Anti-DHFR, C-terminal antibody produced in rabbit D0942-100UG Anti-DHFR, N-terminal antibody produced in rabbit D1067-100UG Order 800-325-3010 Technical Service 800-325-5832 9

FLAG® The octapeptide sequence is highly charged (DYKDDDDK) and useful for sensitive detection, especially if concatenated as the 3xFLAG™ epitope (DYKDHDGDYKDHDIDYKDDDK) . This facilitates the study of low-abundance proteins and the optimization of difficult protein expression projects . It is also popular as a purification tag; the high level of purification can eliminate the need for further clean-up . The FLAG sequence also contains an enterokinase cleavage site to allow removal if the tag is placed at the N-terminus .

There are four types of FLAG antibodies: M1 exhibits Calcium-dependent binding but does not recognize Met-FLAG fusions or unprocessed cytoplasmically-expressed proteins M2 recognizes all types of fusions Polyclonal ANTI-FLAG® recognizes all types of fusions M5 Only recognizes N-terminal Met-FLAG fusions and is not recommended for use in E. coli expression systems

Cat . No . Monoclonal ANTI-FLAG M1 mouse F3040- 2MG. F3040-1MG F3040-5MG Monoclonal ANTI-FLAG M2 mouse purified immunoglobulin F3165- 2MG. F3165-1MG F3165-5MG Monoclonal ANTI-FLAG® M2 mouse Affinity Purified IgG F1804-200UG F1804-1MG F1804-5MG Monoclonal ANTI-FLAG M2-Peroxidase (HRP) antibody produced in mouse A8592- 2MG. A8592-1MG A8592-531MG biomapping

Cat . No . Monoclonal ANTI-FLAG M2-Alkaline Phosphatase antibody produced in mouse A9469- 2MG. A9469-1MG A9469-531MG Monoclonal ANTI-FLAG M2-Cy™3 antibody produced in mouse A9594- 2MG. A9594-1MG A9594-531MG Monoclonal ANTI-FLAG M2-Biotin antibody produced in mouse F9291- 2MG. F9291-1MG F9291-531MG Monoclonal ANTI-FLAG M2-FITC Conjugate antibody produced in mouse F4049- 2MG. F4049-1MG F4049-531MG Polyclonal ANTI-FLAG antibody produced in rabbit F7425- 2MG. Monoclonal ANTI-FLAG M5 antibody produced in mouse F4042- 2MG. F4042-1MG F4042-5MG Monoclonal ANTI-FLAG M5-Biotin antibody produced in mouse F2922- 2MG. ANTI-FLAG M2 Affinity Gel A2220-1ML A2220-5ML A2220-10ML A2220-25ML EZview™ Red ANTI-FLAG M2 Affinity Gel F2426-1ML F2426-531ML Order 800-325-3010 Technical Service 800-325-5832 11

Cat . No . ANTI-FLAG M1 Agarose Affinity Gel A4596-1ML A4596-5ML A4596-10ML A4596-25ML FLAG® Immunoprecipitation Kit FLAG IPT1-1KT ANTI-FLAG® High Sensitivity, M2 coated 96-well plates P2983-1EA FLAG Peptide F3290-4MG F3290-25MG 3x FLAG™ Peptide F4799-4MG F4799-25MG Amino-terminal Met-FLAG-BAP™ Fusion Protein P5975- .1MG Carboxy-terminal FLAG-BAP Fusion Protein P7457- .1MG Amino-terminal FLAG-BAP Fusion Protein P7582- .1MG

Glutathione S-Transferase (GST) GST was one of the first epitope tags to be used; it can be placed on the N or C-terminus and can enhance the solubility of expressed proteins . Purification is achieved with glutathione-conjugated resin .

Cat . No . Anti-GST peroxidase conjugate produced in rabbit A7340- .5ML Monoclonal Anti-GST antibody produced in mouse G1160- .2ML G1160- .5ML biomapping

Cat . No . Glutathione Agarose G4510-1ML G4510-5ML G4510-10ML G4510-50ML Pre-packed columns (3 3 2 .5 mL) G3907-1SET EZview™ Red Glutathione Affinity Resin E6406-1ML E6406-5X1ML

Green Fluorescent Protein (GFP) Unique among epitope tags, GFP is an autofluorescent 27 kDa tag that can be directly detected in living cells by fluorescent microscopy .

Cat. No. Monoclonal Anti-Green Fluorescent Protein (GFP) antibody produced in mouse G6539- 2ML. G6539- 5ML. Monoclonal Anti-GFP, N-terminal antibody produced in mouse G6795-200UG

Anti-GFP, N-terminal antibody produced in rabbit G1544-100UG

Hemagglutinin A (HA) Derived from the binding domain of the Influenza hemagglutinin protein, HA contains a high proportion of charged residues (YPYDVPDYA) so it is likely to form a strong antibody recognition site .

Cat. No. Monoclonal Anti-HA antibody produced in mouse H3663-200UL Monoclonal Anti-HA Peroxidase antibody produced in mouse H6533-1VL (vial of 0 .5 mL) Order 800-325-3010 Technical Service 800-325-5832 13

Cat. No. Monoclonal Anti-HA Alkaline Phosphatase antibody produced in mouse A5477-500UG Monoclonal Anti-HA FITC antibody produced in mouse H7411-100UG Monoclonal Anti-HA TRITC antibody produced in mouse H9037-200UG Monoclonal Anti-HA-Biotin antibody produced in mouse B9183-100UG Monoclonal Anti-HA Agarose antibody produced in mouse A2095-1ML EZview™ Red Anti-HA Affinity Gel E6779-1ML E6779-531ML Anti-HA Immunoprecipitation Kit IP0010-1KT

Histidine (His) This is by far the most widely used purification tag; it allows purification with economical nickel affinity resins . These resins tolerate denaturing conditions (useful for purifying solubilized proteins from inclusion bodies) and can be reused . Binding specificity is less than that of antibody-based resins, so a second purification step is often necessary; this also removes the high imidazole concentration used for elution . Acidic elution has been used as a low-salt alternative to imidazole, and cobalt can been used in place of nickel to increase specificity . Metalloproteins and histidine-rich proteins (e .g ., Chloramphenicol Acetyltransferase) also bind to these resins, so it is advisible to use suitable controls .

Cat. No. Monoclonal Anti-polyhistidine Alkaline Phosphatase antibody produced in mouse A5588- .5ML Monoclonal Anti-polyhistidine Peroxidase antibody produced in mouse A7058-1VL (vial of 0 .5 mL) Monoclonal Anti-polyhistidine antibody produced in mouse H1029- .2ML H1029- .5ML biomapping

Cat. No. HIS-Select® Nickel Affinity Gel P6611-5ML P6611-25ML P6611-100ML P6611-500ML HIS-Select Cobalt Affinity Gel H8162-5ML H8162-25ML H8162-100ML HIS-Select HF Nickel Affinity Gel H0537-10ML H0537-25ML H0537-100ML H0537-500ML EZview™ Red HIS-Select HC Nickel Affinity Gel E3528-1ML E3528-531ML HIS-Select Spin Columns H7787-10EA H7787-50EA HIS-Select® High Capacity (HC) Nickel Coated Plates S5563-1EA HIS-Select High Sensitivity (HS) Nickel Coated Plates S5688-1EA HIS-Select Wash & Elution Buffer Kit H5288-500ML H5413-250ML Order 800-325-3010 Technical Service 800-325-5832 15

Herpes Simplex Virus (HSV) HSV is derived from the glycoprotein D precursor envelope protein and is short (QPELAPEDPED), so it is unlikely to interfere with protein structure or function .

Cat. No. Anti-HSV antibody produced in rabbit H6030-200UG Anti-HSV Peroxidase antibody produced in rabbit H0912-200UL HSV Peptide H4640-4MG H4640-25MG

Luciferase This is better known as a reporter gene for expression assays . Antibodies allow results to be verified by immunological methods .

Cat. No. Monoclonal Anti-Luciferase antibody produced in mouse L2164- .2ML

Maltose-Binding Protein (MBP) The size (43 kDa) of this tag contributes to its ability to increase soluble protein production in E. coli . Along with GST and Thioredoxin it is popular as a ‘solubility’ tag . Purification is achieved using low-cost amylose resin, an Asn10 spacer between the tag and the protein improving resin binding .

Cat. No. Monoclonal Anti-Maltose Binding Protein antibody produced in mouse M6295- .2ML M6295- .5ML Monoclonal Anti-Maltose Binding Protein Alkaline Phosphatase antibody produced in mouse A3963- 5ML. Monoclonal Anti-Maltose Binding Protein Peroxidase antibody produced in mouse A4213-1VL (vial of 0 .5 mL) biomapping

c-Myc c-Myc (or myc) has been extensively used for Western blotting, immunoprecipitation, and flow cytometry . Its small size (EQKLISEEDL) means that it is unlikely to perturb (or enhance) protein folding and it has been used at both N and C-termini .

Cat. No. Monoclonal Anti-c-Myc antibody produced in mouse M4439-100UL Anti-c-Myc antibody produced in rabbit C3956- .2MG Anti-c-Myc Peroxidase antibody produced in rabbit A5598-500UG Monoclonal Anti-c-Myc Biotin antibody produced in mouse B7554-100UG Monoclonal Anti-c-Myc FITC antibody produced in mouse F2047-100UG Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit A7470-1ML EZview™ Red Anti-c-Myc Affinity Gel E6654-1ML E6654-531ML Anti-c-Myc Immunoprecipitation Kit IP0020-1KT c-Myc Peptide M2435-4MG M2435-25MG

Protein A and Protein G These bacterial ‘superantigens’ bind to all forms of IgG . They can be fused to proteins for purification using IgG, but they are most often employed as conjugates for generic secondary detection or purification of antibodies .

Cat. No. Protein A Agarose P2545-1ML P2545-5ML Order 800-325-3010 Technical Service 800-325-5832 17

Cat. No. EZview™ Red Protein A Affinity Gel P6486-1ML P6486-5X1ML EZview Red Protein G Affinity Gel E3403-1ML E3403-5X1ML Protein G Immunoprecipitation Kit IP50-1KT

Streptavidin/Biotin The affinity of the streptavidin-biotin interaction (10-14 M) means that it can withstand harsh conditions, so it is popular as a method of immobilizing proteins (e .g ., in production of protein arrays) . Streptavidin can be incorporated as a full-length, truncated, or mutated protein, and biotin can be incorporated by fusing a protein domain that becomes biotinylated in vivo (e .g . biotin-carboxy carrier protein) .

Cat. No. Anti-Streptavidin antibody produced in rabbit S6390-1ML Monoclonal Anti-Biotin antibody produced in mouse B7653- 2ML. B7653- 5ML. Anti-Biotin antibody produced in goat B3640-1MG Streptavidin−Peroxidase from Streptomyces avidinii S5512-250UG S5512- .5MG S5512-2MG Anti-Biotin−Peroxidase antibody produced in goat A4541- 25ML. A4541- 5ML. A4541-1ML Monoclonal Anti-Biotin−Peroxidase antibody produced in mouse A0185-1VL (vial of 0 .5 mL) Streptavidin−Peroxidase Polymer, Ultrasensitive S2438-250UG biomapping

Cat. No. Streptavidin−Alkaline Phosphatase from Streptomyces avidinii S2890-250UG S2890-1MG Anti-Biotin−Alkaline Phosphatase antibody produced in goat A7064- 25ML. A7064- 5ML. A7064-1ML Monoclonal Anti-Biotin–Alkaline Phosphatase antibody produced in mouse A6561- 2ML. A6561- 5ML. Monoclonal Anti-Biotin–Cy™3 antibody produced in mouse C5585- 2ML. C5585- 5ML. Streptavidin−Cy3 from Streptomyces avidinii S6402-1ML Streptavidin−Gold from Streptomyces avidinii S9059- .4ML S9059-2ML Streptavidin−FITC from Streptomyces avidinii S3762- .1MG S3762- .5MG S3762-1MG Anti-Biotin−FITC antibody produced in goat F6762- .5ML Monoclonal Anti-Biotin–FITC antibody produced in mouse F4024- .2ML F4024- .5ML Biotin-4-Fluorescein B9431-5MG Streptavidin−Fluorescent Polymer, Ultrasensitive S2313-250UG Streptavidin-R-Phycoerythrin from Streptomyces avidinii S3402-1ML Order 800-325-3010 Technical Service 800-325-5832 19

Cat. No. Streptavidin−Agarose from Streptomyces avidinii S1638-1ML S1638-5ML Streptavidin, immobilized on Agarose CL-4B 85881-1ML 85881-5ML EZview™ Red Streptavidin Affinity Gel E5529-1ML E5529-531ML Biotin−Agarose B0519-5ML B0519-25ML Streptavidin−Iron Oxide Particles from Streptomyces avidinii S2415-10ML Streptavidin from Strepotmyces avidinii recombinant S0677-1MG S0677-5MG S0677-25MG Biotin powder, ≥99% B4639-1G B4639-5G More tools for Streptavidin/Biotin are available . Search for ‘Streptavidin’ or ‘Biotin’ on our website at sigma-aldrich.com

T7 This is a 260-residue tag derived from the gene 10 product of T7 phage; may enhance expression levels in E. coli.

Cat. No. Monoclonal Anti-T7 tag antibody produced in mouse T8823-200UG Monoclonal Anti-T7 tag, Peroxidase conjugate antibody produced in mouse T3699-1VL biomapping

Thioredoxin Despite its small size (11 kDa), thioredoxin is very effective as a ‘solubilizing’ tag (As with all solubilizing tags, solubility does not necessarily mean functional folding, and fusion proteins can precipitate when their tag is cleaved) . Thioredoxin is unique among epitope tags in being stable up to 80 °C and can confer some thermostability onto its fusion partner . This way, cellular proteins can be heat-denatured without affecting the fusion protein . Purification can either be achieved using phenylarsine oxide resin or antibody- conjugated resin .

Cat. No. Anti-Thioredoxin antibody produced in rabbit T0803- .2ML T0803- .5ML Anti-Thioredoxin–Agarose antibody produced in rabbit A2582-1ML Thioredoxin from Escherichia coli T0910-1MG

V5 A small tag (GKPIPNPLLGLDST) derived from residues 95-108 of the P/V proteins of the Paramyxovirus SV5 . Vectors, purification resins, and antibodies are widely available .

Cat. No. Monoclonal Anti-V5-Peroxidase antibody produced in mouse V2260-1VL (vial of 0 .5 mL) Anti-V5 antibody produced in rabbit V8137- .2MG Monoclonal Anti-V5 antibody produced in mouse V8012-50UG Monoclonal Anti-V5-Cy™3 antibody produced in mouse V4014-100UG Anti-V5 Agarose Affinity Gel antibody produced in mouse A7345-1ML V5 Peptide V7754-4MG Order 800-325-3010 Technical Service 800-325-5832 21

Vesicular Stomatitis Virus Glycoprotein (VSV-G) The VSV-G antibody recognizes the five C-terminal residues of the tag (YTDIEMNRLGK) . Like many tags, it is found as a cassette in commercially available vectors to aid cloning .

Cat. No. Monoclonal Anti-VSV-G Peroxidase antibody produced in mouse A5977-1VL Anti-VSV-G antibody produced in rabbit V4888-200UG Monoclonal Anti-VSV-Glycoprotein Agarose antibody produced in mouse A1970-1ML VSV-G Peptide V7887-4MG V7887-25MG

Yeast 2-hybrid tags: B42, GAL4, LexA, VP16 These tags are used to detect protein interactions in the yeast 2-hybrid system acting as DNA binding (GAL4, LexA) domains or transcriptional activators (B42, GAL4, VP16) . Antibodies against these tags allow results to be validated and investigated further .

Cat. No. Anti-B42 antibody produced in rabbit B9808-200UG Anti-GAL4, Activation domain antibody produced in rabbit G9293-200UG Anti-GAL4 DNA-BD antibody produced in rabbit G3042-200UG Anti-Lex A antibody produced in rabbit L0415-100UG Anti-VP16 antibody produced in rabbit V4388-200UL biomapping

Further Reading Einhauer, A; Jungbauer, A J . (2001) “The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins ”. Biochem Biophys Methods, Oct 30, 49 (1–3): 455–465 . Makrides, SC . (1996) “Strategies for achieving high-level expression of genes in Eschericia coli ”. Microbiol Rev., Sep, 60 (3), 512–538 . Sheibani, n . (1999) “Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins ”. Prep Biochem Biotechnol., Feb, 29 (1), 77–90 . Stevens, RC . (2000) “Design of high-throughput methods of protein production for structural biology ”. RC Structure, Sep 15, 8 (9), R177–85 . Terpe, K . (2003) “Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems ”. Appl Microbiol Biotechnol., Jan, 60 (5), 523–533 . Waugh, DS . (2005) “Making the most of affinity tags ”. Trends Biotechnol., Jun, 23 (6), 316–20 . Order 800-325-3010 Technical Service 800-325-5832 23

Protocols This section outlines some of the commonly used protocols in protein extraction, purification and detection .

Protein Extraction In order to purify proteins they must be released from the cell and solubilized . Unfortunately, this also releases proteases, so inhibitors must be added to limit proteolysis . Three popular methods for protein extraction are sonication, freeze-thawing, and detergent extraction . All three can be used with any type of starting material, but the most common applications are used here as examples .

Sonication (Escherichia coli) Sonication is achieved by sending high frequency sound waves through a cell suspension . The shear forces induced by the sound waves disrupt solid matter, breaking down tissues and cells . Large masses of cells can be processed, but the procedure is fairly hands-on so it is best suited to small numbers of medium or large scale preparations .

Reagents

Sonication buffer Tris-HCl, pH 7 5. 50 mM NaCl 50–200 mM Reducing agent 5–10 mM Note: Most reducing agents are incompatible with nickel purification resins .

Protease Inhibitor Cocktails Cat. No. General Use, lyophilized powder P2714-1BTL Bacterial, lyophilized powder P8465-5ML P8465-25ML EDTA-Free, solution in DMSO P8849-1ML P8849-5ML

To view more protease and phosphatase inhibitor cocktails, visit sigma.com/protinhib biomapping

Procedure

1 . Harvest cells by centrifugation (3,000 3 g for 15 minutes) .

2 . Resuspend in 1–4 volumes (mass:volume) of ice-cold sonication buffer .

3 . Incubate on ice for 10 minutes . Add protease inhibitor cocktail and mix thoroughly . Note: Some protease inhibitors (e .g ., PMSF) have 4˚C short half-lives in aqueous solution, so it is important to add them fresh .

4 . Keeping the cell suspension on ice, sonicate in 10 second bursts, allowing 20 second cooling period in between . Ten repeats should be sufficient . Note: Try to minimize foaming as this can denature protein . Sonicator horn can shatter glass if it comes into direct contact with it .

5 . Pellet cell debris by centrifugation (20,000 3 g for 20 minutes) . Collect and retain soluble fraction . Note: If the protein is expressed in inclusion bodies then the insoluble fraction should be retained and solubilized in a denaturing buffer, e .g ., denaturing equilibration buffer for histidine purification described later . Order 800-325-3010 Technical Service 800-325-5832 25

Freeze-thawing (cultured cells) Freeze-thawing uses the growth of ice crystals to puncture and break apart cells . It is ideal for studying low-abundance proteins due to the high concentration of sample . First published by Rudolph et al . in 1999 (Anal Biochem 269, 66–71) .

Reagents

Lysis buffer Tris-HCl, pH 7 8. 20 mM KCl 600 mM Glycerol 20%

Procedure

1 . Aspirate medium and wash in ice-cold PBS .

2 . Scrape cells in a minimal volume of ice-cold PBS and transfer to a microcentrifuge tube .

3 . Pellet cells by centrifugation (10,000 3 g for 30 seconds) and carefully discard the supernatant .

4 . Resuspend in lysis buffer (50 μL per 10 cm plate) .

5 . Flash freeze in liquid nitrogen . biomapping

4˚C 6 . Thaw on ice and repeat twice .

7 . Add 250 units of Benzonase® (Cat. No. E1013) and incubate at room temperature for 10 minutes . Note: Benzonase reduces the viscosity of the lysate by digesting genomic DNA, making the sample easier to pipette and pass over chromatography media .

Detergent Extraction (S. cerevisiae) Detergent extraction is suitable for all scales of extraction and circumvents the need for enzymic lysis when used with Saccharomyces cerevisiae . All steps are performed at 4 °C .

Reagents

Cat. No. CelLytic™ Y Cell Lysis Reagent C4482-50ML C4482-500ML Yeast Protease Inhibitor Cocktail, DMSO solution P8215-5ML P8215-25ML To view our full line of lysis reagents, visit sigma.com/lysis

Procedure

1 . Harvest cells by centrifugation (3,000 3 g for 5 minutes) andresuspend in lysis buffer (2 .5–5 mL per gram of cell paste) Note: Addition of 5–10 mM DTT will increase protein yield, especially if cells were harvested after log phase .

2 . Incubate with gentle shaking for 15–30 minutes . Order 800-325-3010 Technical Service 800-325-5832 27

3 . Pellet cell debris by centrifugation (12,000 3 g for 20 minutes) and retain supernatant .

Protein Purification Traditionally, proteins were purified by chromatography based on their native properties (e .g ., size, charge, and hydrophobicity); however, epitope tags allow any protein to be purified by affinity chromatography, which simplifies the procedure and can yield pure preparations after one round of purification . The two most popular types of affinity purification are those employing metal affinity resins (for histidine tags) and antibody-resin conjugates (often termed immunoprecipitation) .

Histidine-tagged Protein Purification Histidine-tagged proteins are purified using the affinity of histidine repeats for nickel ions (Figure 1) This protocol is for small-scale preparations using HIS-Select® Spin Columns (Cat. No. H7787) but large scale preparations can be performed using affinity gel (Cat. No. P6611) . Histidine tags can sometimes be hidden by protein folding, preventing binding to the resin . Because nickel resin is compatible with denaturing agents, this can sometimes be solved by purifying under denaturing conditions; simply solubilize the protein in 8 M urea (Cat. No. U1250) during extraction and use the denaturing buffer recipes . Bear in mind that it is difficult to functionally refold denatured proteins .

Reagents Equilibration Buffer Native Denaturing Sodium phosphate, pH 8 .0 50 mM 50 mM Sodium chloride 0 .3 M — Urea — 8 M Wash Buffer Sodium phosphate, pH 8 .0 50 mM 50 mM Imidazole 5 mM 5 mM Sodium chloride 0 .3 M — Urea — 8 M Elution Buffer Sodium phosphate, pH 8 .0 50 mM 50 mM Imidazole 250 mM 250 mM Sodium chloride 0 .3 M — Urea — 8 M biomapping

Note: Any buffers containing urea must be made fresh daily . The elution buffer pH may need to be varied between 4 .5–6 .0 (depending on the protein), because some recombinant proteins with histidine tags will not elute in the pH 5 .0 to 6 .0 range . If the tagged recombinant proteins will not elute in this range, try a pH as low as 4 .5 . Note: The equilibration and washbuffers should be supplemented with 1–10 mM imidazole and 0 15–0. .5 M sodium chloride to reduce non-specific protein binding . Due to the unique selectivity of the chelate, 5 mM imidazole in the wash buffer is sufficient to obtain high purity samples . Consult the reagent compatibility chart for the use of other reagents .

Procedure

1 . Place HIS-Select® Spin Column in a collection tube .

2 . Add 600 μL of equilibration buffer to the spin column .

3 . Close lid onto spin column and centrifuge at 1,000 rpm (82 3 g) at room temperature for 1 minute . Note: HIS-Select Spin Columns may also be used with an appropriate vacuum manifold in place of the centrifugation step .

4 . Remove spin column from collection tube .

5 . Empty the collection tube and replace the spin column . Order 800-325-3010 Technical Service 800-325-5832 29

6 . Centrifuge prepared cell extract through the column 600 μL at a time (82 3 g for 1 minute) .

7 . Remove spin column from collection tube . Remove and retain the flow-through for later analysis (if required) . Note: Presence of expressed protein could indicate a problem with binding to the resin .

8 . Using a new collection tube, wash unbound protein from the spin column with 600 μL of wash buffer (82 3 g for1 minute) . Remove and retain flow through for analysis (if required) . Note: Presence of expressed protein could indicate problems with resin binding or wash buffer conditions .

9 . Repeat wash step with 600 μL of wash buffer .

10 . Using new collection tube, elute the target protein using up to 500 μL of elution buffer (82 3 g for 1 minute) . biomapping

Reagent Compatibility Chart Compound Max conc . Comments Imidazole, histidine 10 mM Compete with histidine for nickel binding . High concentrations reduce yield Chelating agents (e .g ., EDTA, EGTA) None Can remove nickel ions from the resin Glycerol 50% Helps stabilize proteins 2-mercaptoethanol 20 mM Only use in extraction buffer . Can reduce nickel ions Other reducing agents None Not recommended Nonionic detergents (e .g ., TWEEN®) 2% Glycine None Binds to Nickel; use histidine or Imidazole instead

Tips: • Non-specific binding can be reduced by increasing the imidazole concentration in the wash buffer . • Immunoblotting the crude lysate can be used to verify protein expression . • Use denaturing conditions if you suspect the tag is buried (e .g ., if there is poor recovery despite strong expression) . • Glycerol, salt, detergents, and reducing agents can help reduce protein precipitation . • Retain all fractions for PAGE analysis: it makes problems easier to diagnose and solve . Immunoprecipitation Immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting is routinely used in a variety of applications: • Studying protein/protein interactions • Determining the molecular masses of protein antigens • Monitoring post-translational modifications It also enables concentration and detection of rare proteins which otherwise would be difficult to detect . The technique is essentially small-scale affinity purification; protein is purified on an agarose-linked antibody complex . Agarose is insoluble, so all of the antibody-binding partners can be separated from solution by centrifugation . Many agarose conjugates are available commercially (especially those linked to antibodies against epitope tags), but superantigen (Protein A, G, or L) conjugates are also available, which allow indirect immunoprecipitation if an antibody against the target protein is added first . The choice of superantigen conjugate depends on the species origin and isotype of the primary antibody (Table 2, see page 31) . Order 800-325-3010 Technical Service 800-325-5832 31

Affinity Spectra of Superantigens

Species Immunoglobulin Protein A Protein G Protein L Human IgG (normal) ++++ ++++ ++++ IgG1 ++++ ++++ ++++ IgG2 ++++ ++++ ++++ IgG3 - ++++ ++++ IgG4 ++++ ++++ ++++ IgM - - ++++ IgA - - ++++ IgE - - ++++ IgD - - ++++ Fab ++ ++ ++++ K light chains - - ++++ L light chains - - - ScFv ++ - ++++ Mouse IgG1 + ++++ ++++ IgG2a ++++ ++++ ++++ IgG2b +++ +++ ++++ IgG3 ++ +++ ++++ Rat IgG1 - + ++++ IgG2a - ++++ ++++ IgG2b - ++ ++++ IgG2c + ++ ++++ Bovine IgG ++ ++++ - Cat IgG ++++ - N/A Chicken IgG - + ++ Dog IgG ++++ ++++ + Goat IgG +/- ++ - Guinea Pig IgG ++++ ++ ++ Hamster IgG + ++ ++++ Horse IgG ++ ++++ +/- Pig IgG +++ +++ ++++ Rabbit IgG ++++ +++ + Sheep IgG +/- ++ - Table 2 biomapping

Reagents Antibody-agarose conjugate or primary antibody and superantigen conjugate, e .g ., anti-c Myc agarose (Cat. No. A7470) and protein G agarose (Cat. No. E3403) HNTG buffer Washing buffer (Ice cold): HEPES, pH 7 .5 20 mM HNTG buffer NaCl 150 mM or PBS, pH 7 .4 (Cat . No . P4417) Triton® X-100 0 .1% (w/v) or other buffers e .g ., RIPA buffer Glycerol 10% (w/v) Note: Perform all steps using microcentrifuge tubes on ice unless noted otherwise . Direct Immunoprecipitation (microcolumn) This procedure is taken from the protocol of anti-c-Myc agarose (Cat. No. A7470) . Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin .

1 . Pipette 40–100 µL of the 50% suspension of resin into a microcentrifuge tube or spin-column .

2 . Centrifuge at full speed for 10 seconds in a microcentrifuge . Discard liquid .

3 . Wash the resin 5 times with 1 mL of PBS, using centrifugation to pellet the resin .

4 . Add clarified bacterial lysate or cell extract to the resin pellet .

5 . Bring the volume to at least 200 µL with PBS or RIPA buffer, if needed . Order 800-325-3010 Technical Service 800-325-5832 33

6 . Incubate for 1 .5 hours on a rotator at room temperature or at 4 °C .

7 . Wash the resin 4 times with 1 mL of PBS or lysis buffer .

8 . After the final wash, aspirate the supernatant and leave ~10 µL above the beads .

9 . Centrifuge at full speed for 10 seconds in a microcentrifuge . Discard liquid .

Large scale (5 mL) procedure Immunoprecipitation can also be used for affinity purification on larger scales . This procedure is taken from the protocol of anti-c-Myc agarose (Cat. No. A7470) but can be adapted for other resins .

1 . Place an empty chromatography column, e .g ., 10 mL gravity column (Cat. No. 54806), on a firm support and rinse with PBS .

2 . Allow the buffer to drain from the column, leaving residual PBS in the column to aid in packing the resin .

3 . Thoroughly suspend the vial of resin to make a uniform suspension and immediately transfer the desired volume to the column . biomapping

4 . Allow the agarose bed to settle . Do not let it dry out .

5 . Wash with three sequential 5 mL aliquots of 0 .1 M ammonium hydroxide, pH 11–12, followed by three sequential 5 mL aliquots of PBS .

6 . Load the lysate on the column under gravity flow . Note: Depending upon the fusion protein and the flow rate, not all of the protein may bind . Multiple passes over the column or closing the loaded column and incubating it on a rotator for about 1 hour may improve the binding efficiency .

7 . Collect the flow through of unbound protein for analysis .

8 . Wash the column with PBS until the OD280 of the flow through is <0 01.

9 . Elute the bound c-Myc tagged fusion protein from the column with 10 x 1 mL aliquots of 0 1. M ammonium hydroxide at pH 11–12 into vials containing 30–50 µL of 1 N acetic acid for neutralization . Note: The column may lose activity after prolonged exposure to low pH . Order 800-325-3010 Technical Service 800-325-5832 35

Indirect Immunoprecipitation Indirect immunoprecipitation allows a wider range of proteins to be purified; superantigen resins are used to ‘precipitate’ the antibody complexes so all that is required is an antibody against the target protein . Because there are two binding reactions (superantigen resin-antibody and antibody-lysate) there are two variations of this technique: resin first or lysate first .

1 . Wash agarose conjugate twice with washing buffer, centrifuge for 10 seconds at 12,000 3 g at room temperature . Discard supernatant . Note: If agarose conjugate is a powder, reconstitute it with deionized water and allow it to swell for 5 minutes .

2 . Resuspend agarose conjugate in washing buffer (50% suspension) .

‘Resin First’ method

3 . Divide agarose conjugate into aliquots of 50–100 µL (25–50 µL agarose/bed volume) in microcentrifuge tubes .

4 . Add to each tube 10 µL of primary antibody at appropriate dilution (refer to the antibody specifications) .

5 . Incubate for 15–60 minutes at room temperature, gently mixing the sample on rotator .

6 . Centrifuge at 3,000 3 g for 2 minutes at 4 °C and discard supernatant . biomapping

7 . Wash samples at least 3 times with 1 mL of washing buffer, centrifuge at 3,000 3 g for 2 minutes at 4 °C, and discarding supernatant .

8 . Add to each tube 0 1–1. 0. mL of cell lysate .

9 . Incubate for 90 minutes to overnight at 4 °C, gently mixing the sample on a rotator .

10 . Centrifuge at 3,000 3 g for 2 minutes at 4 °C and discard supernatant .

11 . Wash samples at least 4 times with 1 mL of washing buffer, centrifuge at 3,000 3 g for 2 minutes at 4 °C, and discarding supernatant .

‘Lysate First’ method

2 . Resuspend agarose conjugate in washing buffer (50% suspension) . Order 800-325-3010 Technical Service 800-325-5832 37

3 . Take cell lysate sample (0 .1–1 .0 mL) and add 10 µL of antibody at appropriate dilution (refer to product specification) .

4 . Incubate for 90 minutes to overnight at 4 °C, gently mixing the sample on a rotator .

5 . Add 50–100 µL of agarose conjugate suspension (25–50 µL agarose/ bed volume) . Incubate for 15–60 minutes at 4 °C, gently mixing the sample with a rotator .

6 . Centrifuge at 3,000 3 g for 2 minutes at 4 °C and discard supernatant .

7 . Wash samples at least 4 times with 1 mL of washing buffer, centrifuge at 3,000 3 g for 2 minutes at 4 °C, and discard the supernatant . biomapping

Preparation for SDS-PAGE

1 . Resuspend each pellet in 25–100 µL of Laemmli sample buffer to a final concentration of 13 sample buffer . Heat samples at 95 °C for 5 minutes .

2 . Centrifuge for 30 seconds at 12,000 3 g at room temperature .

3 . Collect supernatant . Samples can be loaded straight away or stored in sample buffer at –70 °C .

Immunoblotting (Western Blotting) Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting . It catalyzes the oxidation of luminol or TMB (3,3′,5,5′‑Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) . Chemiluminescent detection is many times more sensitive than colorimetric, but is more expensive and laborious . Colorimetric detection can be performed on the bench and allows signal development to be observed directly . Immunodetection can be performed directly or indirectly . Direct detection employs HRP-conjugated antibodies to the protein or tag; whereas, indirect is performed in two stages; using an unlabeled ‘primary’ antibody against the protein or tag, and a labeled ‘secondary’ antibody against the first . Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins . The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat. No. A7058 and Cat. No. H1029) and will give results for a wide spectrum of other antibodies . However, optimal results are achieved if the protocol specific to the antibody is followed . Order 800-325-3010 Technical Service 800-325-5832 39

Reagents Transfer Buffer 1 Tris-HCl, pH 10 .4 0 .3 M Methanol 20% Transfer buffer 2 Tris-HCl, pH 10 .4 0 .025 M Methanol 20% Transfer buffer 3 Tris-HCl, pH 10 .4 0 .02 M Methanol 20% e-amino caproic acid 25 mg/mL Blotting and detection buffers PBST PBS with 0 .05% TWEEN® 20 (Cat. No. P3563) Ponceau S solution Cat. No. P7170 Chemiluminescent or colorimetric detection reagents Cat. Nos. CPS160 or T0565

Protein transfer 1 . Build the transfer “sandwich” onto the anode(+) plate as follows: • Two sheets blotting paper soaked in Transfer Buffer 1 • One sheet blotting paper soaked in Transfer Buffer 2 • One sheet of PVDF or nitrocellulose membrane pre-wet with deionized water . Note: If using PVDF, pre-soak in 100% methanol for 10 seconds • Protein gel • Three sheets blotting paper soaked with Transfer Buffer 3 -ve

+ve

2 . Transfer at 1 .5 mA/cm2 for 1 5. hours at room temperature . 3 . Remove the membrane from the apparatus and proceed with immunodetection . Optional: Transfer can be verified by staining in Ponceau S solution for 2 minutes, then rinsing with water prior to blocking . biomapping

Direct Detection

1 . Block the membrane using a solution of 5% non-fat dry milk in PBS for at least 60 minutes .

2 . Wash the membrane three times for 5 minutes in PBST .

3 . Incubate the membrane with anti-polyhistidine, peroxidase conjugate (Cat. No. A7058) in PBST and 1% BSA for 2 hours . Note: Antibody dilution is usually 1:1000, but optimal concentration depends on the sample .

4 . Wash the membrane three times for 5 minutes in PBST .

5 . Treat the membrane with a peroxidase substrate and carry out detection . Order 800-325-3010 Technical Service 800-325-5832 41

Indirect Detection

1 . Block the membrane using a solution of 5% non-fat dry milk in PBS for at least 60 minutes .

2 . Wash the membrane three times for 5 minutes in PBST .

3 . Incubate the membrane with anti-polyhistidine antibody (Cat. No. H1029) as the primary antibody in PBS containing 1% BSA for 2 hours . Note: Antibody dilution is usually 1:1000 but optimal concentration depends on the sample .

4 . Wash the membrane three times for 5 minutes in PBST .

5 . Incubate the membrane with anti-mouse IgG peroxidase conjugate (Cat. No. A9917) diluted 1:80,000 in PBST for 60 minutes .

6 . Wash the membrane three times for 5 minutes in PBST .

7 . Treat the membrane with a peroxidase substrate and carry out detection . biomapping

Immunohistochemistry Immunohistochemical staining is a valuable tool for detecting specific antigens in tissues . Antibody reaction is detected enzymatically through use of HRP or alkaline phosphatase-conjugated secondary antibodies . The procedure consists of a number of steps: • Paraffin removal/rehydration • Antigen retrieval (optional, if signal is weak) • Inactivation of peroxidase (if HRP detection used) • Primary Antibody Reaction • Secondary Antibody (or Extravidin) Reaction • Development • Counterstaining

Reagents and Equipment

Formalin-fixed, paraffin-embedded tissue sections 10 mM phosphate buffered saline, pH 7 .4 (PBS) Cat. No. P3813 or P4417-tablet Bovine serum albumin (BSA) Cat. No. A9647 Diluent 1% BSA in PBS Cat. No. P3688 Xylenes Cat. No. 95692 Ethanol absolute Cat. No. E7023

0 .1% Trypsin in PBS, Trypsin tablet in 4 mM CaCl2, Cat. No. T7409 200 mm Tris, pH 7 .7, or 0 .1% Protease in PBS Cat. No. T7168-tablet Cat. No. P5380 Microwave antigen retrieval solution 10 mM sodium citrate Cat. No. C8532, pH 6 .0, with 1 mM EDTA Cat. No. ED4SS, pH 8 .0 Secondary antibody Enzyme substrate For peroxidase: AEC Staining Kit Cat. No. AEC101, DAB Cat. No. D4418 . For alkaline phosphatase: Fast Red TR/Napthol AS-MX Cat. No. F4648 Mayer’s hematoxylin Cat. No. MHS1 Coplin staining jars Cat. No. S5641 Order 800-325-3010 Technical Service 800-325-5832 43

Paraffin Removal/Rehydration

1 . Place the slides in a 56–60 °C oven for 15 minutes . Caution: Oven temperature must not exceed 60 °C .

2 . Transfer to a xylene bath and perform two changes of xylene for 5 minutes .

3 . Shake off excess liquid and rehydrate slides in two changes of fresh absolute ethanol for 3 minutes .

4 . Shake off excess liquid and place slides in fresh 90% ethanol for 3 minutes .

5 . Shake off excess liquid and place slides in fresh 80% ethanol for 3 minutes .

6 . Rinse the slides in gently running tap water for 30 seconds . Note: Avoid a direct jet which may wash off or loosen the section .

7 . Place in PBS wash bath for further rehydration (30 minutes at room temperature) . biomapping

Antigen Retrieval (optional step for weak signal) Protein epitopes are less accessible in fixed tissues than in solution, so occasionally an antigen “unmasking” step by enzyme digestion may be required . Note: This step is performed only in cases where weak or no staining occurs, or for antigens requiring “unmasking” according to the primary antibody specifications . There are several possible ways for retrieval depending on the antibody and the antigen:

Enzymatic method

1 . Apply 0 .1% trypsin in PBS or 0 .1% protease in PBS for 2–30 minutes at 37 °C . Note: Extending the incubation time may also enhance specific staining .

2 . Rinse in PBS for 10 minutes .

Microwave retrieval

1 . Wash the slides with deionized H2O and place them in a microwave- resistant plastic staining jar containing antigen retrieval solution . Note: Make sure slides are fully covered with solution .

2 . Operate the microwave oven for 5 minutes on high power (~700 watts) . Note: Make sure slides are still covered with retrieval solution or add fresh solution and repeat microwaving .

3 . This process can be repeated 2–3 times .

4 . Let cool slowly at room temperature for at least 20 minutes before proceeding to the next step . Order 800-325-3010 Technical Service 800-325-5832 45

Inactivation of Peroxidase (if HRP detection used) Note: This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase .

1 . Place the slides on a flat level surface . Note: Do not allow slides to touch each other or dry out at any time .

2 . Add drops of 3% hydrogen peroxide to cover the whole section .

3 . Incubate for 5 minutes at room temperature .

4 . Rinse with PBS from a wash bottle .

5 . Place the slide in PBS wash bath for 2 minutes . biomapping

Primary Antibody Reaction Pre-incubation of the sample with 5% BSA for 10 minutes prior to the primary antibody reaction may decrease background staining . For best results with animal tissues, use 5–10% normal serum from the same species as the host of the secondary antibody . Optimal dilution and incubation times should be determined for each primary antibody prior to use .

1 . Allow the slides to drain, shake off excess fluid, and carefully wipe each slide, avoiding the sections .

2 . Dilute the primary antibody or negative control reagent in diluent . Note: The diluent alone may be used as a negative control . A positive control slide (a tissue known to contain the antigen under study) should also be run .

3 . Apply 100 µL of primary antibody solution to the appropriate slides, covering the tissue sections .

4 . Tilt each slide in two different directions, so the liquid is spread evenly .

5 . Incubate for at least 60 minutes at 37 °C in humidified chamber . Note: Longer incubations are advised for low density antigens .

6 . Rinse gently with PBS from a wash bottle . Place the slide in a PBS wash bath for 5 minutes . Order 800-325-3010 Technical Service 800-325-5832 47

Secondary Reaction Option 1: Biotin/Streptavidin Detection

1 . Allow the slides to drain, shake off excess fluid, and carefully wipe the slide as before .

2 . Dilute the biotinylated secondary antibody in diluent to its optimal concentration .

3 . Apply 100 µL to each slide, covering the tissue sections .

4 . Tilt each slide in two different directions .

5 . Incubate in a humidity chamber for at least 30 minutes at room temperature .

6 . Rinse gently with PBS from a wash bottle .

7 . Place the slide in a PBS wash bath for 5 minutes .

8 . Allow the slides to drain, shake off excess fluid, and carefully wipe the slide as before . biomapping

9 . Dilute Streptavidin/ExtrAvidin® conjugate in diluent to its optimal concentration .

10 . Apply 100 µL to all slides, covering the sections .

11 . Tilt each slide in two different directions .

12 . Incubate in humidified chamber for at least 20 minutes at room temperature .

13 . Rinse gently with PBS from a wash bottle .

14 . Place the slide in PBS wash bath for 5 minutes . Note: It is recommended to prepare the detection substrate mixture during this final wash step .

15 . Skip to Development step . Order 800-325-3010 Technical Service 800-325-5832 49

Option 2: Enzyme-labeled Secondary Antibody

1 . Allow the slide to drain, shake off excess fluid, and carefully wipe the slide as before .

2 . Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent .

3 . Apply 100 µL to all slides, covering the tissue sections .

4 . Tilt each slide in two different directions .

5 . Incubate 30 minutes at room temperature or at 37 °C in humidified chamber .

6 . Rinse gently with PBS from a wash bottle .

7 . Place the slides in a PBS wash bath for 5 minutes . Note: It is recommended to prepare the detection substrate mixture during the final wash step . biomapping

Development Note: Addition of 1 mM levamisole (Cat. No. L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme) .

1 . Allow each slide to drain, shake off excess fluid, and carefully wipe the slide as before .

2 . Apply enough drops of freshly prepared substrate mixture to cover the tissue section .

3 . Incubate for 5–10 minutes or until desired color reaction is observed when monitored with the microscope . Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle .

Counterstaining Note: When using AEC substrate, do not use alcohol-containing solutions for counterstaining (e .g ., Harris’ hematoxylin, acid alcohol), since the AEC stain formed by this method is soluble in organic solvents . The slide must not be dehydrated, brought back to toluene (or xylene), or mounted in toluene-containing mountants .

1 . Apply enough Mayer’s hematoxylin to cover the section, or place the slide in a bath of Mayer’s hematoxylin .

2 . Incubate for 30 seconds to 5 minutes, depending on strength of the hematoxylin . Order 800-325-3010 Technical Service 800-325-5832 51

3 . Rinse the slide gently with distilled water from a wash bottle .

4 . Rinse the slide under gently running tap water for 5 minutes Note: Avoid a direct jet, which may wash off or loosen the section .

5 . Mount the sections using an aqueous mounting medium such as glycerol gelatin . Coverslip may be sealed with clear nail polish .

Immunofluorescence Immunostaining is a very versatile technique and, if the antigen is highly localized, it can detect as few as a thousand antigen molecules in a cell . In some circumstances, it can also be used to determine the approximate concentration of an antigen, especially by an image analyzer . As a first step, cells to be stained are attached to a solid support to allow easy handling in subsequent procedures . This can be achieved by several methods: adherent cells may be grown on microscope slides, coverslips, or an optically suitable plastic support . Suspension cells can be centrifuged onto glass slides, bound to solid support using chemical linkers, or in some cases handled in suspension . The second step is to fix and permeabilize the cells, to ensure free access of the antibody to its antigen . Perfect fixation would immobilize the antigens while retaining authentic cellular and subcellular architecture, permitting unhindered access of antibodies to all cells and subcellular compartments . The correct choice of method will depend on the nature of the antigen being examined and on the properties of the antibody used, but fixation methods fall generally into two classes: organic solvents and crosslinking reagents . Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture . Crosslinking reagents (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens . Crosslinkers preserve cell structure better than organic solvents, but may reduce the antigenicity of some cell components, and require the addition of a permeabilization step, to allow access of the antibody to the specimen . Either method may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining . Four different fixation methods are described; the appropriate fixation method should be chosen according to the relevant application . biomapping

The third step of cell staining involves incubation of cell preparations with the antibody . Unbound antibody is removed by washing, and the bound antibody is detected either directly (if the primary antibody is labeled) or indirectly using a fluorophore-labeled secondary reagent . Finally, the staining is evaluated using fluorescence microscopy .

Reagents and Equipment

PBS 0 .01 M Phosphate buffered saline, pH 7 .2–7 .4, Cat. No. P3813 or P4417 Methanol, cooled at –20 °C for at least 1 hour Cat. No. 32213 Acetone, cooled at –20 °C for at least1 hour Cat. No. 24201 3–4% Paraformaldehyde in PBS with 0 .5% Triton® X-100 Cat. No. P6148 Dissolved in PBS using 5 M in PBS NaOH and mild heating Cat. No. T9284 3–4% Paraformaldehyde in PBS Methanol, cooled at –20 °C Cat. No. P6148 Dissolved in PBS, using 5 M for at least 1 hour NaOH and mild heating Cat. No. 32213. PEM Buffer: Ethanol, cooled at –20 °C for at least 1 hour 0 .1 M PIPES with 5 mM EGTA and

2 mM MgCl2 · 6 H2O . Bring to pH 6 .8, using NaOH solution . Cat. No. 270741 Primary antibody Antibody controls Secondary antibody-fluorophore-labeled Aqueous mounting medium Microscope glass slides 76 x 25 mm Cat. No. S8902 Fluorescence microscope equipped with appropriate filter for fluorescence detection Inverted light microscope

Notes: This is provided as a general protocol . Optimal dilutions for the primary and secondary antibodies, cell preparation, controls, as well as incubation times will need to be determined empirically and may require extensive titration . Ideally, one would use the primary antibody as recommended in the product data sheet . The appropriate negative and positive controls should also be included . Protect fluorescent conjugates and labeled slides from the light . Incubate samples in the dark and cover whenever possible . Order 800-325-3010 Technical Service 800-325-5832 53

Cell Preparation 1 . Remove cells from incubator . Inspect under inverted light microscope to verify the desired appearance . Discard medium . 2 . Rinse with PBS, remove excess solution .

Fixation Four different fixation methods are described . Choose the appropriate fixation method according to the application (or product data sheet recommendation) .

Methanol-Acetone Fixation

1 . Fix in cooled methanol, 10 minutes at –20 °C

2 . Remove excess methanol .

3 . Permeabilize with cooled acetone for 1 minutes at –20 °C .

Paraformaldehyde-Triton® Fixation

1 . Fix in 3–4% paraformaldehyde for 10–20 minutes .

2 . Rinse briefly with PBS .

3 . Permeabilize with 0 .5% Triton X-100 for 2–10 minutes . biomapping

Paraformaldehyde-Methanol Fixation

1 . Fix in 3–4% paraformaldehyde for 10–20 minutes .

2 . Rinse briefly with PBS .

3 . Permeabilize with cooled methanol for 5–10 minutes at –20 °C . PEM-Ethanol Fixation

1 . Fix in PEM buffer for 10 minutes .

2 . Rinse twice briefly with PBS .

3 . Permeabilize with cooled ethanol for 5–10 minutes at –20 °C .

4 . Wash three times (at least 5 minutes each) with PBS . Order 800-325-3010 Technical Service 800-325-5832 55

Application of Primary Antibody

1 . Dilute primary antibody in PBS to appropriate dilution . Apply on coverslips over the cells and incubate for 60 minutes at room temperature . Note: A humidified chamber is recommended .

2 . Wash three times for at least 5 minutes with PBS .

Application of Secondary Antibody Note: Secondary antibody is applied only in indirect assays .

1 . Dilute labeled secondary antibody to appropriate dilution in PBS . Apply on coverslips and incubate for 30 minutes at room temperature .

2 . Wash three times (at least 5 minutes each) with PBS .

3 . Remove excess PBS .

Evaluation It is advisable to run the appropriate negative controls . Negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies . The ideal negative control reagent is a fluorophore-conjugated mouse monoclonal or myeloma protein . It should be isotype- matched, not specific for cells of the species being studied, and of the same concentration as the test antibody . The degree of autofluorescence or negative control reagent fluorescence will vary with the type of cells under study and the sensitivity of the instrument used . For fluorescent analysis of cells with Fc receptors, the use of isotype-matched negative controls is mandatory .

1 . Mount coverslips with mounting medium and invert onto glass slides . 2 . Inspect under the microscope and photograph . biomapping

ELISA Enzyme-Linked Immunosorbent Assay (ELISA) is a method for specific quantitation of compounds . Its specificity, flexibility, and reproducibility (as well as low cost) have lead to its widespread use as a diagnostic tool . In a protein research setting, it acts as a convenient method for screening levels of cellular proteins, e .g ., expression profiling . Indirect ELISA Reagents and Equipment

Phosphate buffered saline (PBS) tablet 10 mM phosphate buffer, pH 7 .4, 150 mM NaCl and 0 .1% sodium azide

Carbonate-Bicarbonate buffer capsule, pH 9 .6 (Cat. No. C3041)

Washing buffer (PBST) 10 mM phosphate buffer pH 7 4,. 150 mM NaCl, 0 .05% TWEEN® 20 (Cat. No. P3563)

Antibody controls Species and isotype-matched, non-specific immunoglobulin (e .g ., mouse myeloma proteins as a control for a mouse monoclonal primary antibody)

Secondary antibody Either alkaline phosphatase or horseradish peroxidase (HRP)-conjugate

Detection substrate Alkaline phosphatase: SIGMAFAST™ pNPP tablets (Cat. No. N1891) HRP: SIGMAFAST™ OPD tablets (Cat. No. P9187)

Stopping reagent (optional) Alkaline phosphatase: 3 M NaOH

HRP: 3 M HCl or 3 M H2SO4 Multiwell plates (Cat. No. CLS9017) Order 800-325-3010 Technical Service 800-325-5832 57

Antigen Coating

1 . Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or PBS .

2 . Pipette 0 .2 mL of the above solution into each well of the multiwell plate .

3 . Incubate at 37 °C for 30 minutes, or incubate (covered) overnight at 4 °C .

4 . Remove the coating solution . Wash three times with PBST .

Note: If problems with non-specific binding occur, an additional blocking step (30 minutes with 5% BSA-PBS) may be required. For further information see: Vogt, R .F ., et al., (1987)J. Immunol. Meth., 101, 43 . biomapping

Primary Antibody Reaction

1 . Dilute the monoclonal primary antibody in PBST . The optimal dilution should be determined using a titration assay .

2 . Add 0 2. mL of the diluted monoclonal antibody to each well . The negative control should be species and isotype-matched, non- specific immunoglobulin diluted in PBST .

3 . Incubate at room temperature for 2 hours .

4 . Wash as in step 4 of Antigen Coating .

Application of Secondary Antibody

1 . Dilute the enzyme-conjugated secondary antibody in PBST . Add 0 .2 mL of this solution to each well . The optimal dilution should be determined using a titration assay .

2 . Incubate at room temperature for 2 hours .

3 . Wash as in step 4 of Antigen Coating .

Note: During the last incubation and immediately before use, prepare the enzyme substrate or bring the pre-made liquid substrate to room temperature . Order 800-325-3010 Technical Service 800-325-5832 59

Development

1 . Add 0 2. mL of the freshly prepared substrate to each well .

2 . Color should develop in positive wells after 30 minutes (yellow and orange for pNPP and OPD, respectively) Note: Absorbance may be read directly in a multiwell platereader (at 405 nm and 450 nm for pNPP and OPD, respectively) or the reaction may be stopped with 50 µl per well of the appropriate stopping reagent and absorbance read later .

References

1 . Beesley, J .E . (ed .), (1993) “Immunocytochemistry: A Practical Approach,” IRL Press, 215, 21(Prod . No . Z35,005-2) . 2 . Bullock, G .R . and Petrusz, P . (eds .), (1982), (1983), (1985), (1989) “Techniques in Immunocytochemistry,” Volumes 1, 2, 3 and 4, Academic Press, See Vol . 1, 18 . 3 . Coligan, J .E . et al . (eds .), (1991) “Current Protocols in Immunology ”. John Wiley & Sons, (New York), 2 .1 .1 . 4 . Crowther, J .R ., (1995) “Methods in Molecular Biology, Volume 42: ELISA, Theory and Practice,” Humana Press (New Jersey), (Prod . No . Z36,415-0) . 5 . Cuello, A .C . (ed .), (1993) “Immunohistochemistry II,” Wiley Press, (New York) (Prod . No . I4018) . 6 . Desphande, S .S ., (1996) “Enzyme Immunoassays: From Concept to Product Development,” Chapman & Hall, (New York) (Prod . No . Z37,025-8) . 7 . Diamandis, E .P . and Christopoulos, T .K . (eds .) (1996) “Immunoassay,” Academic Press, (New York), (Prod . No . Z37,367-2) . 8 . Ferencik, M ., (1993) “Handbook of Immunochemistry,” Chapman & Hall, (New York), 346–356 (Prod . No . Z37,020-7) . 9 . Giloh H . and Sedat, J .W . (1982) “Fluorescence Microscopy: Reduced Photobleaching of Rhodamine and Fluorescein Protein Conjugates by n-Propyl Gallate ”. Science, 217, 1252–1255 . 10 . Harlow, E . and Lane, D ., (1988) “Antibodies: A Laboratory Manual ”. Cold Spring Harbor Laboratory Press, (New York), 579 (Cat. No. A2926) . 11 . Johnson, G .D . et al ., (1982) “Fading of Immunofluorescence During Microscopy: A Study of the Phenomenon and Its Remedy ”. J. Immunol. Methods, 55, 231–242 . 12 . Longin A ., et al ., (1993) “Comparison of Anti-Fading Agents Used in Fluorescence Microscopy: Image Analysis and Laser Confocal Microscopy Study ”. J. Histochem. Cytochem, 41(12), 1833–1840 . 13. P. Tijssen, (1985) “Laboratory Techniques in Biochemistry and Molecular Biology: Practice and Theory of Enzyme Immunoassay ”. Elsevier Science Publishers, (Amsterdam, The Netherlands) . 14 . Storz, H . and Jelke, E ., (1984) “Photomicrography of Weakly Fluorescent Objects—Employment of p-phenylenediamine as a Blocker of Fading ”. Acta Histochem., 75, 133–139 . 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