How would you actually measure this property mass you

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Unformatted text preview: iltration? 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. 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). Elution Profile 10 kDa 30 kDa 50 kDa 70 kDa 140 100 kDa 120 Activity (U) 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 90 Fraction Number 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. 100 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: Fractions 49 MW Standards 50 51 52 100 80 70 50 30 10 How could you reconcile the difference between the molecular mass obtained by gel filtration with the one determined by SDS-PAGE? A single band appears in each fraction with a molecular mass of ~25 kDa. However, the gel filtration column suggests that the protein, under native, non-denaturing, conditions has a mass of 50 kDa. Therefore, it is likely that the activity we isolated in fractions 49-51 results from a dimeric complex between two subunits of mass 25 kDa. Note, the two subunits can be identical polypeptides (i.e. our protein is a homodimer) or different polypeptides that coincidentally have the same molecular mass (i.e. a heterodimer). What if, instead of the gel shown in (f), you obtained the following pattern? Fractions MW Standards 49 50 51 52 100 80 70 50 30 10 What would you conclude about the protein’s organization given this gel? Two distinct bands appear in eac...
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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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