UMD BCHM 465 - Exam #2 (10 pages)

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Exam #2



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Exam #2

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Pages:
10
School:
University of Maryland, College Park
Course:
Bchm 465 - Biochemistry III
Biochemistry III Documents

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Biochemistry 465 Section I and only April 15 1999 Exam 2 Prof Jason D Kahn Your Printed Name Your SS Your Signature You have 80 minutes for this exam Exams written in pencil or erasable ink will not be re graded under any circumstances Explanations should be concise You will not need a calculator for this exam and no other study aids or materials are permitted Useful equation P P K 2 BCHM465 Exam 2 4 15 99 1 20 pts Footprinting and protein DNA binding The autoradiogram on the left below shows DNAse I footprinting of a sequence specific DNA binding protein to a labeled restriction fragment The four lanes at the right show increasing total protein concentrations with each lane having about 5 times as much DNA as the one to its left Some of the data on the binding curve at the right came from the experiment on the left o te in sing P r In crea Answer for a 1 00 Binding Curve Fractional Saturation No Protein No DNAse a 6 pts Indicate in the box in the center below where sites A and B are on the gel Explain your reasoning briefly 0 75 0 50 0 25 Site A Site B 0 00 10 10 M 9 10 M 8 7 10 M 10 M Total Protein 6 10 M 5 10 M Note that the gel lanes do not necessarily correspond to particular points on the curves above 1 2 3 4 5 6 b 4 pts Using the binding curve above determine numerical values for the protein s dissociation constants Kd s from sites A and B BCHM465 Exam 2 4 15 99 3 c 4 pts How would the curves above change qualitatively which direction would they shift if the experiment were done at higher salt concentration Why d 6 pts The gel mobility shift or EMSA experiment is often used very qualitatively to identify the presence of a low abundance sequence specific DNA binding protein in a complicated mixture like a nuclear extract Suggest why the EMSA is a better assay for this purpose than a DNAse I footprint hint there s very little of the protein of interest in the mix Why is it often necessary to include a source of non specific DNA e g poly d A T in the reaction



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