One-step analysis of protein complexes in microliters of cell lysate
2005
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. Cells convey signals through cascades of enzymatic reactions and the formation and dissipation of molecular complexes. Many interactions in signal transduction depend on post-translational modifications, complicating the molecular screening of drugs that disrupt signaling complexes. We here demonstrate the testing of inhibitors of signaling-dependent protein complexes in microliters of crude cell lysate using fluorescence (cross)correlation spectroscopy (FCS, FCCS; Fig. 1a,b). In contrast to existing protocols, such as copurification, FCS and FCCS offer (i) separation-free measurements, (ii) the use of only microliters of sample and (iii) high sensitivity at nanomolar probe concentrations. With very few exceptions 1 , previous applications of FCS and FCCS for protein complex analysis used fluorescent fusion proteins or chemical labeling, thereby requiring a dedicated experimental design and restricting detection to selected interactions. Our aim was to make the benefits of FCS and FCCS accessible for a versatile analysis of complexes of endogenous, unlabeled proteins in crude cell lysates. We opted for indirect immunolabeling using matched pairs of primary antibodies against the interaction partners and labeled secondary reagents. We added all reagents in one step to
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