Figure 6. Affinity measurements using a SA sensor chip.

A. Flow cell 1 was loaded with 450 RUs of Biotin-KB-801 peptide, and flow cell 2 was loaded with 350 RUs mNotch peptide as a control. Increasing concentrations of Gα_i1 in GDP+Mg²⁺+AlF₄⁻ (AMF) Buffer, the transition state mimetic form, were injected over the sensor surface as indicated, using the KINJECT command (300 μl injections with a 200 second dissociation phase at 20 μl/min flow rate). Binding curves were obtained by subtracting non-specific binding to a non-interacting biotinylated peptide from all experiment-containing flow cells. B. Binding affinities were determined by plotting the maximum response attained at each concentration of Gα_i1 versus the concentration of the Gα_i1, then by fitting to a rectangular hyperbola to determine K_D using GraphPad Prism 5.0 (Graphpad Software, La Jolla, CA). C. Flow cell 1 was loaded with 450 RUs of Biotin-Gα_i1, and flow cell 2 was loaded with an equivalent RU signal of denatured Biotin-Gα_i1. Varying concentrations of RGS10 in GDP+Mg²⁺+AlF₄⁻ (AMF) Buffer were injected over the sensor surface as indicated, using the KINJECT command (300 μl injections with a 200 second dissociation phase at 20 μl/min flow rate). Specific binding was determined by subtracting non-specific binding from a flow cell containing denatured biotin-Gα_i1. D. Binding affinities were determined by plotting the maximum response attained at each concentration of RGS10 versus the concentration of the RGS10, then by fitting to a rectangular hyperbola to determine K_D using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA). These final panels were reproduced from Soundararajan et al. 2008 (ref. (7)). Copyright (c) 2008 National Academy of Sciences, U.S.A.