Westen Blot problem

[Bioc]

Hi, need a little help here. If you are assaying the pressence of a protein during several stages of development of an animal, and you see that, let's say, P1 and P8 have two bands (let's say P0 is the embryonic stage and no protein iband is seen; and the latter stages are P1 to P9) and the rest (P2 to P7 and P9) one band. What is the most probable explanation to this? And how can you verify it?

This is what I think: 1)the antibody is recognizing more than one epitope (not very specific) or 2) there is a protein with a very similar epitope (not really think is probable, also relates to 1) in some stages or 3) the protein is an oligomer and it is cleaved in some stages (early and adult).

I think 1) is the most probable and I would verify it isolating the lighter protein and re-doing the western blot. If it's positive, then the antibody is not very specific.

Any help would be apreciated, hope you can understand me.

[jimmydasaint]

Hi, need a little help here. If you are assaying the pressence of a protein during several stages of development of an animal, and you see that, let's say, P1 and P8 have two bands (let's say P0 is the embryonic stage and no protein iband is seen; and the latter stages are P1 to P9) and the rest (P2 to P7 and P9) one band. What is the most probable explanation to this? And how can you verify it?

This is what I think: 1)the antibody is recognizing more than one epitope (not very specific) or 2) there is a protein with a very similar epitope (not really think is probable, also relates to 1) in some stages or 3) the protein is an oligomer and it is cleaved in some stages (early and adult).

I think 1) is the most probable and I would verify it isolating the lighter protein and re-doing the western blot. If it's positive, then the antibody is not very specific.

Any help would be apreciated, hope you can understand me.

 

My research is very rusty and I have to strain my memory to the days when I performed Western Blots. However, if I have understood you properly, it is possible for explanation/hypothesis 1 shows crossreactivity of the antibody to other proteins of roughly the same relative molecular mass. However, it is also possible that some protein degradation may have occurred or for an oligomer to break up during the gel electrophoresis.

 

A possible solution is to ask a friendly laboratory to make monoclonal antibody against P2 and, separately to P8 and then to re-do the blot or immunoprecipitations with these two on a gel.

 

Last time I got two bands instead of one which was expected, I am pretty sure it was down to cross contamination of antigens

[ecoli]

Not sure I understand the P# development stage jargon.

[CharonY]

I do not understand the description either. Do you mean that only at one stage you do not see a band and in all the others you do? Or do you mean you see it in certain stages but in not in subsequent ones? Are you referring to different proteins? What is the lighter protein?

I am pretty sure some crucial information is missing.