scholarly journals Tandem Affinity Purification of Protein Complexes from Mammalian Cells

BioTechniques ◽  
2002 ◽  
Vol 33 (2) ◽  
pp. 267-270 ◽  
Author(s):  
D.M. Cox ◽  
M. Du ◽  
X. Guo ◽  
K.W.M. Siu ◽  
J.C. McDermott
Keyword(s):  
Mammalian Cells ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification

Interaction proteomics: characterization of protein complexes using tandem affinity purification–mass spectrometry

2010 ◽  
Vol 38 (4) ◽  
pp. 883-887 ◽  
Author(s):  
Pamela Völkel ◽  
Perrine Le Faou ◽  
Pierre-Olivier Angrand
Keyword(s):  
Protein Complex ◽  
Mammalian Cells ◽  
Protein Identification ◽  
Biochemical Characterization ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein A ◽  
High Sensitivity ◽  
Tandem Affinity Purification

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP–calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP–MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.

scholarly journals Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

2019 ◽  
Vol 96 (1) ◽  
Author(s):  
Guillaume Adelmant ◽  
Brijesh K. Garg ◽  
Maria Tavares ◽  
Joseph D. Card ◽  
Jarrod A. Marto
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification

scholarly journals Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination

2005 ◽  
Vol 4 (11) ◽  
pp. 1942-1950 ◽  
Author(s):  
Bernd Schimanski ◽  
Tu N. Nguyen ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Affinity Chromatography ◽  
Protein Function ◽  
Protein Identification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Related Factor ◽  
Protease Cleavage ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

scholarly journals Discovery of Protein Complexes with Core-Attachment Structures from Tandem Affinity Purification (TAP) Data

2012 ◽  
Vol 19 (9) ◽  
pp. 1027-1042 ◽  
Author(s):  
Min Wu ◽  
Xiao-li Li ◽  
Chee-Keong Kwoh ◽  
See-Kiong Ng ◽  
Limsoon Wong
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Attachment Structures
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