Please explain part 3. to this question, and explain how you derived your answer
ID: 53060 • Letter: P
Question
Please explain part 3. to this question, and explain how you derived your answer. Thank you very much.
You have produced a monoclonal antibody that binds to the protein actin. To be sure that the antibody does not cross-react with other proteins, you test your antibody in a western blot assay on whole-cell lysates that have been subjected to electrophoresis under nondenaturing conditions (without SDS and Mercaptoethanol, figure A below) and denaturing conditions (B). Does the antibody cross-react with other proteins? If so, does this explain the results in the two western blots? If not, how do you explain the difference observed? List the procedures you would follow to obtain the data in question 1 and 2 above. Highlight how they differ.Explanation / Answer
Here, the antibody is generated against the protein actin.
The monoclonal antibody probably does not croos-react with other proteins; the differential results in Non-denaturing conditions (A) & Denaturing conditions (B) is probably due to the reason :
Actin is a globular protein and so in non-denaturing conditions, actin does not readily migrate towards the anode as it does in denaturing conditions - this is due to the reason that the highly folded structure of actin is preserved in non-denaturing conditions and in denaturing conditions, SDS coats the proteins with negative charge which readily leads to the migration of the proteins toards the anode. So the antibody binds to folded actin in (A) & denatured linearised actin in (B).
The fact that in denaturing conditions, only a single band is obtained proves that the antibody does not croos-react; if it did, then at least two bands would have been obtained, one for actin & other for the protein which cross-reacts with the antibody.