Abstract
We present 'mix and measure' procedures for the analysis of protein complexes in microliters of crude human and mouse cell lysates using fluorescence correlation and crosscorrelation spectroscopy. We labeled interacting endogenous proteins by indirect immunofluorescence with all primary and secondary reagents added in one step. Especially for the screening of compounds interfering with interactions that depend on signaling-induced posttranslational modifications, the approach represents a major advance over existing protocols.
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Acknowledgements
We thank G. Jung for excellent facilities in peptide chemistry and A. Nordheim for constant support. K.K. is a scholar of the Graduiertenkolleg 794. R.B. gratefully acknowledges financial support from the Volkswagen Foundation (“Nachwuchsgruppen an Universitäten”).
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I.C.D.J. is employed by Miltenyi Biotec GmbH, a company that produces some of the reagents used.
Supplementary information
Supplementary Fig. 1
Detection of the interaction of ZAP-70-YFP and CD3ε or ζ. (PDF 114 kb)
Supplementary Fig. 2
Mass tag-mediated detection of the interaction of endogenous ZAP-70 and CD3ε. (PDF 80 kb)
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Stoevesandt, O., Köhler, K., Fischer, R. et al. One-step analysis of protein complexes in microliters of cell lysate. Nat Methods 2, 833–835 (2005). https://doi.org/10.1038/nmeth802
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DOI: https://doi.org/10.1038/nmeth802
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