Question

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 (shown in gel A) and denaturing conditions (shown in gel 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 differences observed?

Answer 1

To determine if the antibody cross-reacts with other proteins, perform a western blot assay using whole cell lysates subjected to both nondenaturing and denaturing conditions. If additional bands are detected in the nondenaturing gel, it indicates cross-reactivity.

Step-by-step explanation:

The antibody should be tested for cross-reactivity with other proteins. This can be done by subjecting whole cell lysates to electrophoresis under both nondenaturing conditions (gel A) and denaturing conditions (gel B) and performing a western blot assay using the monoclonal antibody that binds to actin.

If the antibody cross-reacts with other proteins, it would result in the detection of additional bands in the western blot. The presence of bands in gel A but not gel B would suggest that the antibody binds to other proteins that undergo denaturation during gel B electrophoresis.

On the other hand, if the antibody does not cross-react with other proteins, the differences observed in the western blot results between gel A and gel B could be due to differences in the conformation or accessibility of the actin protein under non-denaturing and denaturing conditions.