Hi Robert, I am not familiar with your assay, but why do you cut the membrane?

If it is because other bands are positive, then I am afraid that you are using a bad antibody.

Plus, if you are using an HRP secondary and chemiluminescence to detect your protein, then a 10 min exposure is far too long.

Do you have a blot of your cells that you only incubate with the secondary Ab and expose for 10 min as a control?

Thanks for the answer. We cut the membrane because we'd rather blot each portion of membrane separately rather than blotting and reblotting and reblotting...About the method, we are using chemoluminiscence (ECL). We did controls with just the secondary and these bands didn't appear.

We think it is something about the protein or the antibody.

Hi Robert, I don't understand your rationale for probing sections of the membrane and I don't understand what you mean by "blotting and reblotting".

Anyway, to me, 10 min exposures using ECL indicates that you have very low protein concentrations in your lysates or you are using a bad primary antibody.

Can you do IP of your lysates to increase the concentrations for the gel?

If your antibody is specific to the phosphorylated version of your protein,  is it possible to add dephosphorylated fractions as a control?  These lanes should have no (or atleast greatly reduced) interaction with your protein of interest and could be used to help you rule out non-specific bands on your gel.  Also, if you want to see if your primary is working properly, I would think you need to blot the whole gel.  For the blot and reblot issue, are you looking for multiple proteins so you would have to strip and reprobe?

Yes, we are studying at the same membrane proteins which weight 36 and 55 kd 

Hallo,

can you please tell me if you still remember. what were these 4 bands. because i have now the same problem yours. with 4 bands.

thanks