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7.06_2004_PS2key - 7.06 Spring 2004 PS 2 KEY 1 of 7 7.06...

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7.06 Spring 2004 PS 2 KEY 1 of 7 7.06 Problem Set 2 Answer Key 1) You have started working in lab that investigates signaling pathways downstream of TGFß receptors. One of the senior graduate students has been working on a screen for novel TGFß’s and has one that she thinks might interest you—she calls it TGFß57. She’s already cloned the gene so you can make pure TGFß57. You decide to take up the challenge of characterizing this new growth factor. a) You would first like to find some high affinity receptors that bind your novel growth factor. Design an experiment to identify the cell-surface TGFß57 receptors. Answer: There is no one right way to do this, however, a common method is to radiolabel the purified growth factor with iodine-125 ( 125 I) and culture the labeled growth factor with cultured cells (you would probably use the same fibroblast cell line that’s been used in earlier experiments with TGFß57). Then you’d treat the culture with a chemical cross- linker to covalently bind the growth factor to any receptors it bound. You can then use standard purification methods to purify the labeled receptors from cell membranes (non- ionic detergents to separate the receptors from the cell membrane and chromatography or electrophoresis to separate your receptor from other proteins). You would now like to investigate the binding properties of TGFß57. b) You decide to look at the maximal physiological response of TGFß57R to TGFß57. Based on the graph below determine the K d (in relative concentration) of TGFß57 binding, the percentage of receptors occupied at 50% of maximal physiological response, and the percentage of maximal physiological response when 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: K d 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.
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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 K d 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 125 I-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
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