Abstract
This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5–2.5 h for data acquisition and analysis.
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Acknowledgements
We thank B. Ramani, Y. Ishitsuka and K. Ragunathan for help with developing the protocol. This work was funded by US National Institutes of Health grants (AI083025, GM065367 to T.H.; HL082846 to Y.K.X.). Additional support was provided by National Science Foundation grants (0646550, 0822613 to T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.
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A.J., Y.K.X. and T.H. designed the research. R.L. prepared the cell extracts. A.J. conducted single-molecule experiments and analyzed the data. A.J., Y.K.X. and T.H. wrote the paper.
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Immunoprecipitation of endogenous proteins from mouse tissues. (DOC 61 kb)
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Jain, A., Liu, R., Xiang, Y. et al. Single-molecule pull-down for studying protein interactions. Nat Protoc 7, 445–452 (2012). https://doi.org/10.1038/nprot.2011.452
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DOI: https://doi.org/10.1038/nprot.2011.452
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