7.06_2004_PS1key - 7.06 Spring 2004 PS 1 key 1 of 11 7.06...

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7.06 Spring 2004 PS 1 key 1 of 11 7.06 Problem Set 1 1. You would like to purify a protein(s) that is responsible for some activity (call it X) in the cell. Fortunately, you have at your disposal a very simple colorimetric assay to monitor the activity you are interested in. a) List at least 3 possible techniques you could use to purify the activity out of a crude cell lysate and the basis by which these methods separate molecules of interest (note: you do not know the identity of the activity producing molecule, so antibodies cannot be used). Method Property 1) Rate-zonal Centrifugation Mass 2) Ion-exchange Chromatography Charge 3) Gel Filtration Chromatography Mass How do you know you have isolated the activity of interest? You need to realize that after fractionation, each sample/fraction must be tested for the activity X by using the “colorimetric assay.” b) You use the colorimetric assay to measure the enzyme’s activity in the crude lysate and found it to be 5000 units. When you examine your fractions, however, none seem to contain any measurable activity. How could this have happened? The enzyme is a multi-subunit complex that requires all subunits for activity – you may have disrupted the complex during purification (i.e. you purified the subunits away from one another). Alternatively, the protein may have precipitated/denatured under your purification conditions. c) As previously mentioned, your lysate contained 5000 units of activity. But, unlike the situation outlined in (b), what if one fraction contains 50, 000 units of activity. Under what circumstances could this happen (i.e. how could you have 10x more activity in your purified fraction than in your total lysate)? The enzyme’s activity in the crude lysate may be restrained by an inhibitor. During purification, you may have separated the two, unleashing the full activity of the enzyme. d) You decide to use Gel Filtration to purify your activity. What property are you exploiting by using gel filtration? How would you actually measure this property? Mass. You must run proteins of known molecular weight through the column and determine in which fractions they elute. You can estimate the size of your protein by comparing the fraction(s) it eluted into with the fractions that the molecular weight standards eluted into.
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7.06 Spring 2004 PS 1 key 2 of 11 e) You perform the gel filtration experiment and measure the activity using your nifty colorimetric assay. The following results are obtained (the elution position of molecular weight standards is superimposed on the profile). What is the estimated molecular mass of the molecules in the sample with the highest activity? Fraction 50 has the highest activity and contains molecules of approximately 50 kDa. Elution Profile 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 70 80 90 100 Fraction Number Activity (U) 70 kDa 100 kDa 50 kDa 10 kDa 30 kDa
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7.06 Spring 2004 PS 1 key 3 of 11 f) However, after performing SDS-PAGE on fractions containing the highest levels of activity and staining the gel with Coomassie Blue (a dye that reveals proteins), you observe the following:
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