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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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.

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


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

  • 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

  • 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

  • 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.
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Afshar, K., Barton, N.R., Hawley, R.S., and Goldstein, L.S.B. 1995 DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin‐like protein. Cell 81:129‐138.
   Brizzard, B.L., Chubet, R.G., and Vizard, D.L. 1994. Immunoaffinity purification of FLAG epitope–tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques 16:730‐734.
   Chaing, C.‐M., Ge, H., Wang, Z., Hoffman, A., and Roeder, R.G. 1993. Unique TATA‐binding protein–containing complexes and cofactors involved in transcription by RNA polymerases II and III. EMBO J. 12:2749‐2762.
   Chang, C.‐P., Brocchieiri, L., Shen, W.‐F., Largman, C., and Cleary, M.L. 1996. Pbx modulation of hox homeodomain amino‐terminal arms establishes different DNA‐binding specificities across the hox locus. Mol. Cell Biol. 16:1734‐1745.
   Chu, W., Burns, D.K., Swerlick, R.A., and Presky, D.H. 1995. Identification and characterization of a novel cytokine‐inducible nuclear protein from human endothelial cells. J. Biol. Chem. 270:10236‐10245.
   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.
   Dent, P., Reardon, D.B., Morrison, D.K., and Sturgill, T.W. 1995. Regulation of raf‐1 and raf‐1 mutants by ras‐dependent and ras‐independent mechanisms in vitro. Mol. Cell Biol. 15:4125‐4135.
   Evan, G., Lewis, G., Ramsay, G., and Bishop, J.M. 1985. Isolation of monoclonal antibodies specific for human c‐myc proto‐oncogene product. Mol. Cell. Biol. 5:3610‐3616.
   Guan, X.‐M., Kobilka, T.S., and Kobilka, B.K. 1992. Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. J. Biol. Chem. 267:21995‐21998.
   Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.P., Urdal, D.L., and Conlon, P.J. 1988. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6:1204‐1210.
   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.
   Lilley, G.G., Dolezal, O., Hillyard, C.J., Bernard, C., and Hudson, P.J. 1994. Recombinant single‐chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV. J. Immunol. Methods 171:211‐226.
   Molloy, S.S., Thomas, L., VanSlyke, J.K., Sternberg, P.E., and Thomas, G. 1994. Intracellular trafficking and activation of the furin proprotein convertase: Localization to the TGN and recycling from the cell surface. EMBO J. 13:18‐33.
   Prickett, K.S., Amberg, D.C., and Hopp, T.P. 1989. A calcium‐dependent antibody for identification and purification of recombinant proteins. BioTechniques 7:580‐589.
   Shelness, G.S., Morris‐Rogers, K.C., and Ingram, M.F. 1994. Apolipoprotein B48–membrane interactions. Absence of transmembrane localization in nonhepatic cells. J. Biol. Chem. 269:9310‐9318.
   Thut, C.J., Chen, J.‐L., Klemm, R., and Tijan, R. 1995. p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Science 267:100‐104.
   Tucker, S.J., Bond, C.T., Herson, P., Pessia, M., and Adelman, J.P. 1996. Inhibitory interactions between two inward rectifier K+ channel subunits mediated by the transmembrane domains. J. Biol. Chem. 271:5866‐5870.
   Wilson, I., Niman, H., Houghten, R., Cherenson, A., Connolly, M., and Lerner, R. 1984. The structure of an antigenic determinant of a protein. Cell 37:767‐778.
   Zhang, X.‐K., Wills, K.N., Husmann, M., Hermann, T., and Pfahl, M. 1991. Novel pathway for thyroid receptor action through interaction with jun and fos oncogene activities. Mol. Cell Biol. 11:6016‐6025.
Key Reference
   Kolodziej and Young, 1991. See above.
  A good general review of epitope tagging.
Internet Resources
  Scientific Imaging Systems web page, which provides further information on anti‐FLAG antibodies and conjugates, FLAG expression vectors, kits, and accessories.
PDF or HTML at Wiley Online Library