Two distinct bands appear in each fraction with

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Unformatted text preview: h fraction with molecular mass of >10 (~15) kDa and >30 (~35) kDa. Therefore, it is likely that the activity we isolated in fractions 49-51 results from a heterodimeric complex of two subunits of mass 35 and 15 kDa. In the gel filtration column, the protein is purified under 7.06 Spring 2004 PS 1 key 4 of 11 native, non-denaturing, conditions and therefore exhibits the molecular mass of the 50 (35 +15) kDa complex. g) After successfully purifying the protein, you intend to determine its identity. List two methods that you could use to identify the protein of interest if you are given a test tube of purified enzyme. 1) Edman Degradation 2) Mass-Spec 2. You have decided to do a UROP and end up working in a lab that studies transport proteins. This doesn’t concern you, however, since you learned all about transport proteins in 7.06. Your graduate student advisor is attempting to create an in vitro system involving cellular Ca2+ flux in order to study Ca2+ triggered intracellular signaling. However, your advisor is busy with other problems and doesn’t have time to choose the best Ca2+ pump for the system. He thus asks you to find the best Ca2+ pump for his system. (a) Being the expert that you are, you know the first thing to do is to collect different kinds of Ca2+ pumps to test. List two methods of enriching for Ca2+ pumps in the membrane. Answer: One approach is to extract and purify a specific transport protein, then reincorporate into a liposome. A second approach is to express the gene encoding the transport protein at high levels in a cell type that doesn’t normally express the protein. (b) Now that you’ve obtained the initial rates of Ca2+ intake by the pumps, you can determine the Vmax and Km of each Ca2+ pump. What does Vmax and Km tell you about a given Ca2+ pump? Answer: The Vmax gives you a measure of the maximal rate of the pump, in other words it tells how well it would transport Ca2+ if every pump was in the process of moving an ion of Ca2+ at every possible moment. The Km tells you ho...
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This note was uploaded on 01/23/2012 for the course LSM lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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