Problem 2. Equilibrium binding, Scatchard plot, Hill plot, K d


Question Description:

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Problem 2. Equilibrium binding, Scatchard plot, Hill plot, Kd and Bmax. The following data are from an experiment measuring equilibrium binding of various concentrations of the same radioligand L to the same 1 ml samples of membranes used in problem 1. Nonspecific binding was determined in the presence of 10 mM unlabeled ligand. Total Ligand Conc. (nM) Total cpm bound Non- specific cpm bound 0.1 15595 833 0.3 46551 2498 0.5 77164 4163 1 151938 8325 3 410233 24975 5 565970 41625 10 700239 83250 Plot specific binding (Total bound – nonspecific bound) against [L*]. Make initial estimates of Kd and Bmax from this graph (2A). Plot the specific binding on a semilog plot (2B) to see if that helps. Construct a Scatchard plot (2C) of the data and derive new estimates of Kd and Bmax. For these calculations consider that free [L*] = counts added/sample – specific cpm bound. Does the Kd from this Scatchard differ from that obtained in plot 2A, and if so, why? How does this Kd compare with that obtained measuring the rates of association and dissociation in Problem 1? Construct a Hill plot (Log [B/(Bmax-B) vs. log[L]). What can you conclude from the slope and intercept of this plot? SHOW YOUR WORK for all your calculations.

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