Epitope Tagging of Recombinant Proteins

Bill Brizzard1, Richard Chubet1

1 Eastman Kodak Company, New Haven, Connecticut
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.8
DOI:  10.1002/0471142301.ns0508s00
Online Posting Date:  May, 2001
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Abstract

Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion gene is cloned into an appropriate expression vector for the experimental cell type and host cells are transfected. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag. This unit presents protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols in this unit employ the anti‐FLAG M2 antibody to detect and purify FLAG‐tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti‐FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti‐FLAG M2 affinity chromatography.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immunoprecipitation of Flag Fusion Proteins from COS Cells Using Anti‐Flag M2 Affinity Gel
  • Basic Protocol 2: Detection of Flag Fusion Proteins by Western Blot
  • Basic Protocol 3: Purification of Flag Fusion Proteins by Anti‐Flag M2 Affinity Chromatography
  • Reagents and solution
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunoprecipitation of Flag Fusion Proteins from COS Cells Using Anti‐Flag M2 Affinity Gel

  Materials
  • COS cells transfected with a FLAG fusion protein using DEAE‐dextran (see CPMB UNIT and appendix 1A in this manual)
  • Anti‐FLAG M2 affinity gel (Eastman Kodak)
  • Lysis buffer (see recipe)
  • Bovine serum albumin (BSA)
  • Tris‐buffered saline (TBS; appendix 2A)/1 mM CaCl 2
  • TBS ( appendix 2A)
  • 2× SDS sample buffer ( appendix 2A)
  • FLAG peptide (Eastman Kodak)
  • 9‐ or 56‐cm2 tissue culture dish
  • Cell scraper
  • Tube rotator
  • Heating block, 100°C
  • Microconcentrator (e.g., Amicron Micron 3; optional)
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; see CPMB UNIT and appendix 1A in this manual)

Basic Protocol 2: Detection of Flag Fusion Proteins by Western Blot

  Materials
  • 2× SDS sample buffer ( appendix 2A)
  • Nitrocellulose membrane
  • TBS ( appendix 2A)
  • Anti‐FLAG BioM2 monoclonal antibody (Eastman Kodak)
  • Streptavidin‐HRPO conjugate
  • Blocking solution: 5% nonfat dry milk in distilled water
  • Enhanced chemiluminescence kit (ECL; Amersham)
  • Additional reagents and equipment for SDS‐PAGE and blotting of SDS‐polyacrylamide gels to nitrocellulose membrane (see CPMB UNITS & and appendix 1A in this manual)

Basic Protocol 3: Purification of Flag Fusion Proteins by Anti‐Flag M2 Affinity Chromatography

  Materials
  • TBS ( appendix 2A)
  • Anti‐FLAG M2 affinity gel (Eastman Kodak)
  • 0.1 M glycine⋅HCl, pH 3.5
  • FLAG peptide (Eastman Kodak)
  • 1 M Tris base (if eluting protein with glycine⋅HCl)
  • TBS ( appendix 2A)/0.02% sodium azide
  • Disposable 10‐ml (0.8 × 4–cm) polypropylene column (Bio‐Rad)
  • Ring stand
  • Tubing, 1.6 mm i.d.
  • Tubing clamp
  • Microconcentrator (Amicon Centricon 10; optional)
NOTE: Preequilibrate the column and perform all steps at room temperature. If desired, column chromatography may also be performed at 4°C to reduce protease activity.NOTE: The anti‐FLAG M2 affinity gel is resistant to the following detergents (not a comprehensive list): 5.0% Tween 20, 5.0% Triton X‐100, 0.1% Nonidet P‐40 (NP‐40), 0.1% CHAPS, and 0.2% digitonin. It can also be used with 1.0 M NaCl or 1.0 M urea. Use of the gel in the presence of SDS, deoxycholate, or guanidine⋅Cl is not recommended.
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Figures

Videos

Literature Cited

Literature Cited
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   Chubet, R.G. and Brizzard, B.L. 1996. Vectors for expression and secretion of FLAG epitope–tagged proteins in mammalian cells. BioTechniques 20:136‐141.
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   Knappik, K. and Pluckthun, A. 1994. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. BioTechniques 17:754‐761.
   Kolodziej, P.A. and Young, R.A. 1991. Epitope tagging and protein surveillance. Methods Enzymol. 194:508‐519.
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Key Reference
   Kolodziej and Young, 1991. See above.
  A good general review of epitope tagging.
Internet Resources
   http://www.kodak.com/daiHome/SIS/ksis.shtml
  Scientific Imaging Systems web page, which provides further information on anti‐FLAG antibodies and conjugates, FLAG expression vectors, kits, and accessories.
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