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要旨:
Obtaining quantitative kinetic parameters from FRAP (Fluorescence Recovery After Photo-bleaching) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries, stripes and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry that creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by more accurately parameterizing the initial post-bleach state. This allows for "reconstruction" of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLCdelta1 in C. elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.
