期刊
NATURE PROTOCOLS
卷 11, 期 2, 页码 316-326出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.020
关键词
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资金
- University of Cambridge
- Cancer Research UK
- European Research Council (ERC)
- European Molecular Biology Organization (EMBO)
- Cancer Research UK [22310, 20411] Funding Source: researchfish
Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.
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