Figure 5. Quantification of the binding affinity by equilibrium pull-down assay.

(A). Representative Coomassie-blue-stained SDS-PAGE gels of the supernatant samples in equilibrium pull down experiments involving GST-Rac1 (Q61L/P29S) binding to ΔWRC230. (B) Schematic representation of the three successful equilibrium models used to fit the binding isotherms. (C) Binding isotherms fit to the indicated models. For WT and C179R ΔWRC230, results for single-site (dotted curves) and two-site (solid curves) models are shown. For Y967A WRC, results for single-site (dotted curves) and GST-dimer models (solid curves) are shown. The data were pooled from multiple independent experiments (5 experiments for WT and C179R, and 2 experiments for Y967A) covering various concentration ranges. Derived dissociation constants are shown in the right table, with K_D,D representing the dissociation constant for the D Site, K_D,A for the A Site, K_D,A(D) for the A Site when the D Site is occupied by Rac1, and K_D,A’ for the A Site of the second WRC in the GST-dimer model. Standard errors of the fit for the derived K_D values are shown. p values were obtained by F-test; the most appropriate dissociation constants based on the p values are shown in bold. See Figure 5—figure supplement 2 and Methods for additional details.

Figure 5—figure supplement 1. Representative Coomassie blue-stained SDS-PAGE gels of the supernatant (top gel) and the beads samples (bottom gel) in an equilibrium pull-down assay involving GST-Rac1 (Q61L/P29S) binding to ΔWRC230.

The top gel is identical to the top gel in Figure 5A. Each reaction contained 30 μL of beads and 70 μL of supernatant. Samples for the top gel (supernatant) were prepared by mixing 40 μL of the supernatant with 8 μL of a 6X SDS PAGE loading buffer, with 15 μL of the samples (thus 12.5 μL of the supernatant) loaded to each lane. Samples for the bottom gel (beads) were prepared by adding 60 μL of a 2X SDS PAGE loading buffer to the remaining 60 μL of reaction (containing 30 μL of supernatant and 30 μL of beads), with 2.5 μL of the mixture (thus 0.625 μL of the supernatant and 0.625 μL of beads) loaded in each lane.

Figure 5—figure supplement 2. Quantification of the binding affinity by equilibrium pull-down assay involving GST-Rac1 (Q61L/P29S) bindingΔWRC230.

(A) Schematic representation of all equilibrium models used to fit the binding isotherms. (B) Binding isotherms fit to the indicated models, with dotted curves for single-site, thick solid curves for two-site sequential, and thin solid lines for GST-dimerization models. The data were pooled from multiple independent experiments (5 experiments for WT and C179R, and 2 experiments for Y967A) covering various concentration ranges. (C) The derived dissociation constant K_D (μM, shown with standard errors of the fit) and the fitting results used for F-test (Df for degree of freedom, RSS for residual sum of squares) are shown in the left table. F-test results are shown on the right with column colors matched to the curve colors in (B) (**** for p<0.0000001). The results for the best fits based on the p values are shown in bold.