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Epitope Tags in Protein Research Tag Selection & Immunotechniques Biomapping

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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 PPrrooteintein 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) EpitopeEpitope tag Proteinotein 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
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