期刊
JOURNAL OF CELL BIOLOGY
卷 183, 期 2, 页码 223-239出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200805092
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资金
- Wellcome Trust Program [073980/Z/03/Z]
- Biotechnology and Biological Sciences Research Council
- Engineering and Physical Sciences Research Council
- Scottish Higher Education Funding Council
- Medical Research Council (MRC)
- Human Frontier Science Program fellowship
- Caledonian Research Foundation fellowship
- MRC
- RASOR
- Biotechnology and Biological Sciences Research Council [BB/C511572/1, BB/C511613/1] Funding Source: researchfish
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.
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