Faculty of Biochemistry, Biophysics and Biotechnology, Division of Cell Biophysics, Jagiellonian University, Kraków, Poland.
Cytometry A. 2010 Apr;77(4):366-70. doi: 10.1002/cyto.a.20866.
Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent way of data analysis.
荧光漂白恢复（FRAP）是一种广泛应用于研究活细胞中荧光标记蛋白动态行为的工具。我们分析了已发表的关于各种核蛋白动力学的数据，并注意到 FRAP 方案和数据分析方法在实验室之间存在差异。一个问题是实验方案是否会影响恢复时间。为了确定 FRAP 方案是否会影响荧光半恢复时间，我们使用了各种 FRAP 方案，并研究了 GFP 标记的 H1（连接）组蛋白的动力学。我们证明荧光半恢复时间取决于漂白方案，包括漂白光的光通量。因此，我们得出结论，由于方案之间以及数据分析方式的差异，关于各种核蛋白流动性的现有信息不允许在来自不同实验室的实验之间进行直接比较。为了充分发挥 FRAP 作为定量技术的潜力，需要为光漂白方案建立基本规则，并采用一致的数据分析方法。
