Question

4. The first stage of western blotting involves separating proteins using ger electrophoresis, On what basis does this technique separate proteins? 5. How can you test the efficiency of transfer of the protein from the gel to the nitrocellulose membrane?

Answer 1

4. The first stage of western blotting invovles seperating proteins using gel electrophoresis. On what basis does this technique seperate ptrotein?

The first stage of wetern blotting is the seperating of protein by gel electrophoresis. SDS-PAGE isthe widely used technique to seperate proteins based on the mass of proteins. SDS-PAGE is a kind of agarose gel electrophoresis and is a discontinuous electrophoretic system. Protein is unfolded to linear form and SDS acts as a detergent which can coat them with overall negative charge. It makes the protein to migrate toward the positive end of the gel and be separated based on their size and their charge. Therfore, smaller proteins move quickly through the gel than larger proteins reslting in formation of band. The size of the protein in the band can be compared with standards of known sizes.

5. How can you test the efficiency of transfer of the protein from the gel to the nitrocellulose membrane?

• Pre stained protein ladder- It can be seen whether brightly colored ladder bands are still in the gel, or have transferred completely to the membrane.
• Stain the gel with Coommassie blue - It has be done once the transfer is complete, Clear gel indicates complete transfer of protein.
• To determine where the duration is too long - insertion of two membrane right behind the other. If the duration is quite long, presence of protein could be detected in second membrane as well and this is more likely to occur to the smallest proteins