Question

parts a,b, c please

3. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis a. After pouring the SDS polyacrylamide gel, you realize that instead of 2 ml, you used 4 ml of 30% acrylamide/bisacrylamide solution for the preparation of the separating gel (in both cases for 5 ml, total gel volume). How would this affect the separation of your proteins during SDS PAGE? Explain your answer! b. You have to remake the gel and are now making sure just to add 2 mL of 30% acrylamide/bisacrylamide solution (in 5 mL total volume) for the separating gel. After you finished preparing both parts of the gel, you double check the pH values of a) the running buffer, b) the separating gel buffer (AKA TRIS buffer for preparation of the separating gel) and c) the stacking gel buffer (AKA TRIS buffer for preparation of the stacking gel). Your measurements are a) 8.5, b) 9, and c) 4. Considering what you learned in this course, how would you expect the resulting gel to look like after Coomassie Staining? Explain! buffer forurter for b) 9. and c. You learned two methods to visualize your proteins after SDS PAGE. Which one would you suggest to use, if you need to determine that your protein was successfully purified? Explain why you chose this method!

Answer 1

3. A) Addition of 2ml 30% acrylamide in 5ml solution has lesser percentage of gel than 4ml of 30% acrylamide in 5ml solution. Incrased volume of acrylamide increases the cross link. So the pore size decreases.

The higher the percentage of gel the lower the separation speed. If the percentage increases, cross links increases. Reduced pore size affects the running of high molecular weight protein in the gel. So the rate of separation is directly affceted by the percentage of separating gel.

B) Rechecked pH of the running buffer is 8.5, pH of the separating gel buffer is 9 and stacking gel buffer is 4.

The actual pH of all the above buffers should be 8.3, 8.8 and 6.8 respectively.

At the pH 6.8 of stacking gel the glycinate ions attain neutral(zwitterion) charge and they gradually move down towards anode and stack the protein into the stacking layer. If the pH is 4 then the glycine attain positive charge and they will not move towards anode. This adversely affect the separation of protein at the stacking gel itself. So you could see no bands in the gel after coomassie staining.

C) I would prefer westerblotting after SDS PAGE. This method is completely based on antigen antibody interaction. So this is highly specific. So accurate prediction of my protein of interest can be done with this technique.