National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Biophys J. 2010 Nov 3;99(9):3093-101. doi: 10.1016/j.bpj.2010.08.059.
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 molecules in FCS is mainly responsible. When this effect is minimized, FRAP and FCS measurements nearly agree, although cross-validation by other approaches is now required to rule out mutual errors. Our results demonstrate the necessity of a photobleach correction for FCS measurements of GFP-tagged molecules that are bound for >0.25 s, and represent an important step forward in establishing a gold standard for in vivo binding measurements.
现在可以在活细胞中定量结合，但这种测量的准确性仍然不确定。为了解决这个不确定性，我们比较了荧光恢复后光漂白（FRAP）和荧光相关光谱（FCS）测量活细胞细胞核中转录因子糖皮质激素受体结合动力学的结果。我们发现，FRAP 测量的结合停留时间比 FCS 测量的结果长 15 倍。我们表明，这种差异不太可能是由于 FRAP 和 FCS 通常使用的浓度有显著差异造成的，也不太可能是由于核的空间异质性、FCS 焦点体积的不当校准、或有意的 FRAP 光漂白造成的。相反，我们的数据表明，FCS 中结合分子的光漂白是主要原因。当这种影响最小化时，FRAP 和 FCS 测量结果几乎一致，尽管现在需要通过其他方法进行交叉验证来排除相互误差。我们的结果表明，对于结合时间大于 0.25 秒的 GFP 标记分子的 FCS 测量，需要进行光漂白校正，这是建立体内结合测量金标准的重要一步。
