I have some questions about Western Blotting:
1. Buffer composition is very important to successful Western Blot. If buffer concentrations of this ingredient are to low your gel swells dramatically resulting in poor resolution while high concentrations desiccate so that protein transfer is poor. What ingredient is it?
A. HCl
B. Tris
C. Methanol
D. SDS
2. What will occur if the relative order of the gel and the membrane in figure 1 are reversed?
A. The temperature will spike and the gel will melt
B. electricity will not flow and the transfer will not occur
C. protein will move out of the gel into the filter paper
D. that would be just fine
3. What will happen if small bubbles are caught between the gel and the membrane?
A. Nothing
B. bubbles interrupt transfer creating holes in image
C. bubbles create hot spots which melt the gel
D. bubbles increase transfer efficiency creating black spots on image
4. Why should you not write on your membrane with pen or sharpie?
A. dyes move with the electrophoresis introducing background
B. membrane will rip and ruin transfer
C. Methanol dissolves the dye introducing background
D. protein will bind to dye interfering with transfer
5. The blocking buffer often contains milk, or bovine serum albumin (BSA). What does it suggest if the background is higher when blocking with milk rather than BSA?
A. Primary or secondary antibody binds a milk protein
B. Primary or secondary antibody binds to BSA protein
C. Transfer was less efficient with milk.
D. Transfer was less efficient with BSA.