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Unformatted text preview: half the receptors are occupied. What does this tell you
about the percentage of bound receptors needed to achieve maximal response?
*** NOTE: y-axis label: Fraction of maximum cellular response Answer: Kd is about 1.2. At half-maximal physiological response, only about 26% of the
receptors are bound. When 50% of receptors are bound, about 77% of the maximal
physiological response is elicited. This tells you that cells do not nee to have 100% of the
receptors bound by ligand in order to achieve a maximal response. 7.06 Spring 2004 PS 2 KEY 2 of 7 c) You would now like to determine the binding affinity of TGFß57 for the TGFß57
receptor that you have been working with. Design an experiment to measure the Kd of TGFß57
binding high- affinity receptors in a cultures fibroblast cell line. Explain how you would deal
with TGFß57 binding nonspecifically to low-affinity receptors.
Answer: You’d want to design some kind of binding assay to measure receptor binding.
What is commonly done is to take a sample of cells and incubate them with increasing
concentrations of 125I-labeled TGFß57. After incubation, you separate the cells from
unbound TGFß57 (usually by centrifugation) and the amount of radioactivity bound to
them is measured. This gives you the total binding of TGFß57 to receptors. In order to
determine specific binding to high-affinity receptors you have to measure nonspecific
binding. This is determined by repeating the binding assay in the presence of a 100-fold
excess of unlabeled TGFß57, which saturates all the specific high-affinity sites. In this case
all the labeled V binds to nonspecific sites. To determine specific binding you simple
calculate the difference...
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- Spring '11