Abstract
Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.
Highlights
Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors
First TATA-binding protein (TBP) is incorporated with 13–14 TBP-associated factors (TAFs) into the TFIID complex involved in pol II transcription [13]
The sample was separated on a 1D gel, digested in gel with trypsin, and analyzed by mass spectrometry (Raw data, available as a Scaffold file, are available upon request.)
Summary
Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. We applied SILAC combined with affinity purification to investigate the specificity of protein complex interactions. For all detected unique TAF peptides the ratio of the signal of their unlabeled version versus their labeled version (SILAC ratio) was determined (A list of all quantified peptides and proteins, including Mascot score, protein name, and IPI accession number, and raw data are available upon request).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
