Analysis of Binding at a Single Spatially Localized Cluster of Binding Sites by Fluorescence Recovery after Photobleaching

摘要: Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a three-dimensional FRAP model that accounts single spatially localized cluster of binding sites the presence both diffusion and impermeable boundaries. We demonstrate completely ignoring spatial localization yield poor estimates parameters site In contrast, find only restricted axial height binding-site is far less detrimental, thereby enabling use computationally expensive models. also identify simplified solutions to limiting behaviors where either or dominate. show how role can sometimes produce serious errors parameter estimation. illustrate application method by analyzing transcription factor, glucocorticoid receptor, tandem array mouse mammary tumor virus promoter cells, obtaining an estimate vivo constant (10(-7) M), first approximation upper bound on transcription-factor residence time at (approximately 170 ms). These analysis tools will important measuring key cellular necessary complete accurate description networks regulate behavior.