Supplementary Figure 11: Data input field of the binding curve analysis (“BC analysis”) sheet.
From: Quantifying macromolecular interactions in living cells using FRET two-hybrid assays
(a) Background subtracted raw data (columns D-H) are automatically adopted from the “Samples” sheet. The principal calculation steps required in order to solve binding curves are automatically performed in columns I-W for each (biological) cell according Steps 117-130 outlined in the Procedure section. (b) The table in the upper left of the worksheet contains all parameters (columns F and I) necessary to correct for spectral properties and spurious FRET. Thereby, all values except the excitation ratio ((ƐYFP(440)/ƐCFP(440)) are automatically transferred from previously analyzed sheets. Values for KdEff, EAmax and EDmax (Column K) are place holders that are adjusted during the fitting procedure. (c) Graphs shown displays data points for individual biological cells (blue dots) and the fitted binding isotherme (black lines). The graphs represent data for 33-FRET (Dfree plotted against EA) and E-FRET (Afree plotted against ED) and allow a control of the fitting procedure.
