Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy.

ABSTRACT: Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin bin

SUBMITTER: Michelman-Ribeiro A 

PROVIDER: S-EPMC2711375 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature