Abstract
Antibody-based affinity purification is a recognized method for use in studying protein–protein interactions. There are four different classes of proteins that are typically identified with such affinity purification workflows: bait protein, proteins that specifically interact with the bait protein, proteins nonspecifically associated with the antibody, and proteins that cross-react with the antibody. Mass spectrometry can be used to differentiate these classes of proteins in affinity-purified mixtures. Here we describe the use of stable isotope labeling by amino acids in cell culture, substrate trapping, and mass spectrometry to enable the objective identification of the components of affinity-purified protein complexes.
Original language | English (US) |
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Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press Inc. |
Pages | 219-234 |
Number of pages | 16 |
DOIs | |
State | Published - 2023 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2603 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Bibliographical note
Funding Information:SNT acknowledges funding from the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and start-up funds from the University of Minnesota Department of Laboratory Medicine and Pathology.
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Keywords
- AAA+ ATPases
- Affinity purification
- Bait protein
- Mass spectrometry
- Protein
- Protein interactions
- SILAC
- Substrate trapping
- Amino Acids/chemistry
- Isotope Labeling/methods
- Proteins/chemistry
- Mass Spectrometry/methods
- Proteomics/methods
- Cell Culture Techniques
PubMed: MeSH publication types
- Research Support, Non-U.S. Gov't
- Journal Article
- Research Support, N.I.H., Extramural