Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates.

ABSTRACT: Binding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS. We show that this discrepancy is not likely due to the significant differences in concentrations typically used for FRAP and FCS, nor is it likely due to spatial heterogeneity of the nucleus, improper calibration of the FCS focal volume, or the intentional FRAP photobleach. Instead, our data indicate that photobleaching of bound mole

SUBMITTER: Stasevich TJ 

PROVIDER: S-EPMC2966010 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature