Werner Stefan, Ebenhan Jan, Haupt Caroline, Bacia Kirsten
Institute of Chemistry, ZIK HALOmem and Charles-Tanford-Protein Center, University of Halle, Kurt-Mothes-Str. 3a, 06120, Halle, Germany.
Chemphyschem. 2018 Dec 19;19(24):3436-3444. doi: 10.1002/cphc.201800576. Epub 2018 Nov 29.
Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows binding analysis of biomolecules. Combining cross- and autocorrelation amplitudes yields binding degrees and concentrations of bound and unbound species. However, non-ideal detection volume overlap reduces the cross-correlation, causing overestimation of the K . The overlap quality factor that relates measured and true cross-correlation amplitudes has been difficult to determine, because neither a perfect 1 : 1 labeled sample nor perfectly overlapping volumes are readily accomplished. Here, we describe how a stochastically labeled sample can be used for quantitative calibration. Lipid vesicles doped with green and red fluorescent dyes yield highly reproducible relative cross-correlations and allow determination of the setup-dependent overlap quality factor. This reliable, affordable and quick-to-prepare calibration standard expedites any quantitative co-localization or binding analysis by dcFCCS.
双色荧光交叉相关光谱技术（dcFCCS）可用于生物分子的结合分析。结合交叉相关和自相关振幅可得出结合程度以及结合态和非结合态物质的浓度。然而，非理想的检测体积重叠会降低交叉相关，导致对解离常数（K）的高估。由于既不容易获得完美的1:1标记样本，也不容易实现完美重叠的体积，因此难以确定将测量的交叉相关振幅与真实交叉相关振幅联系起来的重叠质量因子。在此，我们描述了如何将随机标记的样本用于定量校准。掺杂绿色和红色荧光染料的脂质囊泡可产生高度可重复的相对交叉相关，并可确定与仪器设置相关的重叠质量因子。这种可靠、经济且易于制备的校准标准可加快任何通过dcFCCS进行的定量共定位或结合分析。
