Question

1) Why was the membrane incubated in blocking buffer? What would be the consequence of failing to block the membrane?

2) Explain the process of loading protein ladder and actin/myosin protein onto the gel with the fish samples and how it is necessary for analysis of the gel and immunoblot?

3) Why was a Bradford assay performed with the fish protein extracts? Fully explain why this was necessary for analysis of the gel and immunoblot?

4) The molecular mass of Myosin Light Chain is approximately 22KD, myosin heavy chain is 210KD and actin is 42KD. Which protein will migrate the fastest through the gel and why?

Answer 1

Answer 1. The membrane is incubated in the blocking buffer, so that the non specific binding site of the membrane could be blocked.

Non specific binding side :- this occurs when antibodies are binded to the wrong place which would not have been made for them. (non specific site)

Immunoassay (detecting specific protein through their properties as antigens or antibodies). In immunoassay when we fail to block non specific binding site of the membrane, then antibodies will be binded to the lower affinity target,this may alter the result.