Abstract
This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification. In addition, we use a bicistronic expression system that ensures recovery of stably transfected cell lines expressing easily detectable levels of the protein of interest. A method is also presented for generating cytoplasmic and nuclear extracts, which extends use of this protocol to identify protein–protein interactions occurring specifically in the cytoplasm or nucleus. This protocol facilitates the preparation of partially purified recombinant protein and identification of protein–protein interactions in mammalian cell culture models. The protocol can be completed in 34 h.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Cazalla, D., Sanford, J.R. & Caceres, J.F. A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells. Protein Expr. Purif. 42, 54–58 (2005).
Drakas, R., Prisco, M. & Baserga, R. A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells. Proteomics 5, 132–137 (2005).
Forler, D., et al. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21, 89–92 (2003).
Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).
Rigaut, G., et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).
Geisse, S., Gram, H., Kleuser, B. & Kocher, H.P. Eukaryotic expression systems: a comparison. Protein Expr. Purif. 8, 271–282 (1996).
Bürckstümmer, T. et al. An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat. Methods. 3, 1013–1019 (2006).
Schimanski, B., Nguyen, T.N. & Günzl, A. Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Eukaryot. Cell 4, 1942–1950 (2005).
Liang, S. & Lutz, C.S. p54nrb is a component of the snRNP-free U1A (SF-A) complex that promotes pre-mRNA cleavage during polyadenylation. RNA 12, 111–121 (2006).
Tange, T.O., Shibuya, T., Jurica, M.S. & Moore, M.J. Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core. RNA 11, 1869–1883 (2005).
Gingras, A.-C., Aebersold, R. & Raught, B. Advances in protein complex analysis using mass spectrometry. J. Physiol. 563, 11–21 (2005).
Brajenovic, M., Joberty, G., Kuster, B., Bouwmeester, T. & Drewes, G. Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network. Journal Biol. Chem. 279, 12804–12811 (2004).
Knuesel, M. et al. Identification of novel protein–protein interactions using a versatile mammalian tandem affinity purification expression system. Mol. Cell Proteomics 2, 1225–1233 (2003).
Liu, Z., Cashion, L.M. & Twu, J.J. A systematic comparison of relative promoter/enhancer activities in mammalian cell lines. Anal. Biochem. 246, 150–152 (1997).
Geng, J. & Carstens, R.P. Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures. Protein Expr. Purif. 48, 142–150 (2006).
Jackson, R.J., Howell, M.T. & Kaminski, A. The novel mechanism of initiation of picornavirus RNA translation. Trends Biochem. Sci. 15, 477–483 (1990).
Jang, S.K., Kräusslich, H.G., Nicklin, M.J., Duke, G.M., Palmenberg, A.C. & Wimmer, E. A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636–2643 (1988).
McCracken, S. et al. Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin. J. Biol. Chem. 280, 42227–42236 (2005).
Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).
Kapust, R.B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001).
Blum, H., Beier, H. & Gross, H.J. Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8, 93–99 (1987).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Tsai, A., Carstens, R. An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1, 2820–2827 (2006). https://doi.org/10.1038/nprot.2006.371
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.371
This article is cited by
-
A novel protein purification scheme based on salt inducible self-assembling peptides
Microbial Cell Factories (2023)
-
JMJD6 regulates histone H2A.X phosphorylation and promotes autophagy in triple-negative breast cancer cells via a novel tyrosine kinase activity
Oncogene (2019)
-
The maize brevis plant1 is a type II inositol polyphosphate 5-phosphatase
Journal of Plant Biochemistry and Biotechnology (2017)
-
Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes
Nature Protocols (2016)
-
Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein
Nature Neuroscience (2011)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.