Problem with signal detection

[sharmakaushikp]

I am able to detect signals in all the lanes of my blot using Amersham's ECL Plus detection reagents. but however, when i try using normal ECL reagent (Amersham) for the same blot, i can see signal from the corner bands only and not along the entire lenth of the blot. the protein detected is beta tubulin, to confirm equal loading, which is seen with ECL Plus. i am not able to figure out where the problem lies. please help me out.

[kiki06]

Hi

What membrane type are you using PVDF or other?

Also are you preparing it in any way before western blotting - Methanol? Etc?

Make sure during incubation that the shaker is slowly rocking so that your whole membrane is under the solution and

During ECL incubation, make sure that you have the whole membrane covered with ECL.

Could be a transfer issue, but more likely a ECL / antibody incubation issue.

Let us know which one it is when you solve it.

[sharmakaushikp]

Thanks for the suggestion. I am using nitrocellulose membrane (Amersham).
All antibody incubations were done with sufficient volume of antibody solution, and also sufficient ECL volume was used. Anyway, i will try again.
Thanks again.