A Combined Approach for the Localization and Tandem Affinity Purification of Protein Complexes from Metazoans

2005 ◽  
Vol 2005 (266) ◽  
pp. pl1-pl1 ◽  
Author(s):  
I. M. Cheeseman ◽  
A. Desai
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Combined Approach

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

A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes

PROTEOMICS ◽  
2007 ◽  
Vol 7 (23) ◽  
pp. 4228-4234 ◽  
Author(s):  
Christian Johannes Gloeckner ◽  
Karsten Boldt ◽  
Annette Schumacher ◽  
Ronald Roepman ◽  
Marius Ueffing
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Native Protein

Isolation of protein complexes from the model legumeMedicago truncatulaby tandem affinity purification in hairy root cultures

2016 ◽  
Vol 88 (3) ◽  
pp. 476-489 ◽  
Author(s):  
Jonas Goossens ◽  
Nathan De Geyter ◽  
Alan Walton ◽  
Dominique Eeckhout ◽  
Jan Mertens ◽  
...  
Keyword(s):  
Hairy Root ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Hairy Root Cultures ◽  
Root Cultures
Sign in / Sign up

Export Citation Format

Share Document