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One-step analysis of protein complexes in microliters of cell lysate

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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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Figure 1: Schematic of protein complex analysis by FCCS and FCS using indirect immunolabeling.
Figure 2: Detection of stimulation-dependent protein complexes and testing of inhibitors of complex formation.

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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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Authors and Affiliations

  1. Department of Molecular Biology, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany

    Oda Stoevesandt, Karsten Köhler, Rainer Fischer & Roland Brock

  2. Miltenyi Biotec GmbH, Friedrich Ebert Str. 68, Bergisch Gladbach, 51429, Germany

    Ian C D Johnston

Authors
  1. Oda Stoevesandt
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  2. Karsten Köhler
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  3. Rainer Fischer
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  4. Ian C D Johnston
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  5. Roland Brock
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Correspondence to Roland Brock.

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Competing interests

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)

Supplementary Methods (PDF 121 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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