7. Techniques of protein purification a. Why is SDS omitted when proteins need to undergo isoelectric focusing? b. A series of proteins with a known molecular mass and an enzyme of unknown molecular mass are separated by chromatography on a Sephadex G-200 column. The elution volume (Ve) for each of the protein is indicated in the table below Blue dextran lysozyme Chymotrypsinogen ovalbumin serum albumin aldolase urease ferritin Unknown M 1000 kDa 14 kDa 25 kDa 45 kDa 65 kDa 150 kDa 500 kDa 700 kDa ? Ve (mL) 85.00 200.00 190.00 170.00 150.00 125.00 90.00 92.00 130.00 Estimate the molecular mass of the unknown protein (hint: draw the graph) c. Give a plausible explanation for an unusual behavior of ferritin's elution from the sephadex column. d. A student isolates a protein from anaerobic bacteria and analyses the protein by poly acrylamide gel electrophoresis containing SDS (PAGE-SDS). Following protein staining, a single band appeared. To be certain the supervisor suggests the student run a second electrophoresis under native conditions (i.e.non-denaturing or without SDS). The gel shows two bands after staining. Assuming no errors were committed during these experiments, explain the observations.