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biochemistry study questions

biochemistry study questions - MIT Department of Biology...

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PBC Study Questions (Spring 2005) 1 7.02/10.702 Protein Biochemistry Study Questions Spring 2005 The problems in this packet are taken from exams from the past few semesters. As always, these questions are not meant to be exhaustive, but to give you an idea of what topics to study. We strongly urge you to work through the questions BEFORE looking at the answer key, and to bring any questions to your TAs or Instructors. MIT Department of Biology 7.02 Experimental Biology & Communication, Spring 2005
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PBC Study Questions (Spring 2005) 2 Question 1 You have a crude lysate sample (CL) containing a mixture of six proteins (1, 2, 3, 4, 5, and β - galactosidase), and your goal is to obtain purified β -galactosidase. Some characteristics of these proteins are shown in the table below: Protein Concentration of ammonium sulfate (AS) required for precipitation Molecular Weight (kDa) Isoelectric point (pI) 1 45% 38 3.7 2 80% 22 4.8 3 65% 4 5.3 4 20% 75 6.8 5 30% 55 9.50 β -galactosidase 45% 115 5.3 You begin your purification by performing an ammonium sulfate (AS) precipitation. You add the appropriate concentration of AS to your CL sample, incubate overnight at 4˚C, then centrifuge to generate a supernatant (AS-S) and pellet (AS-P). a) What concentration of AS will you use to precipitate β -galactosidase? _______ b) After addition of that concentration of AS and centrifugation, which protein(s) will be in the supernatant (AS-S)? ________________________ c) Which protein(s) will be in the pellet (AS-P)? __________________ d) After resuspending the AS-P in column buffer, you should use a PD-10 column to _____________ your sample. One way to purify β -galactosidase away from any contaminating proteins in the AS-P sample would be to separate the proteins based on their molecular weight. e) What type of column separates on this basis? _________________ f) Which protein (from your AS-P) would elute first from this type of column? _______ Instead, you decide to use ion exchange chromatography to further purify β -galactosidase away from other proteins in your AS-P sample. You first run an anion exchange column equilibrated using column buffer with a pH of 5.0. g) What charge does the matrix of an anion exchange column have? _________ h) At pH 5.0, which protein(s) from the AS-P will stick to the column? _________ i) Explain your answer to part i) in one or two sentences.
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PBC Study Questions (Spring 2005) 3 Question 1 (continued) j) State how you would elute a protein bound to an anion exchange column, and explain how this elution method works in one or two sentences . You identify the fraction containing β -gal from your anion exchange column, and decide to run it over a cation exchange column to complete your purification. k) Describe how you would use a cation exchange column to purify β -galactosidase away from any remaining contaminating protein(s). Be specific about: 1) the pH at which you’d equilibrate the column; 2) why you chose this pH; and 3) which protein(s) would bind and which protein(s) would flow through the column under these conditions, and why.
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