Nagy Péter, Vereb György, Damjanovich Sándor, Mátyus László, Szöllõsi János
University of Debrecen, Debrecen, Hungary.
Curr Protoc Cytom. 2006 Nov;Chapter 12:Unit12.8. doi: 10.1002/0471142956.cy1208s38.
This unit presents protocols describing the measurement of protein associations using FRET as determined by flow and image cytometry. The proteins under investigation can be labeled by fluorescent antibodies or fluorescent protein (FP) variants. The flow cytometry protocols determine FRET based on the measurement of donor quenching, which provides a FRET value on a population basis, or based on the measurement of fluorescence intensities in the donor, FRET, and acceptor channels, which provides cell-by-cell FRET values. An extension of this protocol is based on cell-by-cell correction for autofluorescence and requires the measurement of four fluorescence intensities. The algorithm described can be applied in image cytometric FRET as well. The image protocol determines FRET resolved by donor photobleaching. The authors provide extensive discussion of pitfalls, limitations, and interpretation.
本单元介绍了使用流式细胞术和成像细胞术测定蛋白质相互作用的实验方案。所研究的蛋白质可用荧光抗体或荧光蛋白（FP）变体进行标记。流式细胞术实验方案基于供体淬灭的测量来确定FRET，它提供群体水平的FRET值，或者基于供体、FRET和受体通道中荧光强度的测量，从而提供逐个细胞的FRET值。该实验方案的一个扩展基于逐个细胞的自发荧光校正，需要测量四种荧光强度。所描述的算法也可应用于成像细胞术FRET。成像实验方案通过供体光漂白来确定FRET。作者对实验中的陷阱、局限性及解释进行了广泛讨论。
