Everytime i try to develop my film to see if the bands appear on the film it either turns out blank or jus shows an ouline of the membrane.
ive thought it may be the ECL but i did the blue test for that and it was glowing and i have also tested the film which is fine.
The developer and fixer are fine too as some1 else used it and gained results.
The transfer also worked as i stained it with Coomassie blue and mostly all d proteins were transferred and the rainbow marker was present on the membrane after the transfer.
And i dont think its due to antibody degradation since not even the rainbow marker is showing up on the film.

Anybody got any ideas??? i need to write this up in my final year project and i havent come up with a conclusion 2 why this is happening! pleaseee help!!!!

daisy

Daisy - are you using a secondary (e.g. anti-mouse-HRP) to detect? If so, you could try troubleshooting by using "Magic-Mark" markers from Invitrogen (or something simlilar) - this should stain with your secondary and give you bands on your film.

If this works, then it would seem to be a problem with you primary antibody.

Let us know how you progress!

PW

i am using antigoat(rabbit) as my secondary antibody.
i dont think there is a problem with my antibodies as the molecular markers also dont show up on the film.
The project has already been completed as it is a final year project which lasts only 5weeks. i need to know why this is happening so i can write it up in my thesis. iv tried to think of everthing but i dont know why its not showing up on the film.

THanks for ur help!!!
any more ideas??? lolz

[quote=daisy]i am using antigoat(rabbit) as my secondary antibody.
i dont think there is a problem with my antibodies as the molecular markers also dont show up on the film.

Your problem lies in one of two places -

1. your primary antibody has gone bad or doesnt react on western blots.

2. your secondary antibody is not working properly - you can test this by
A. dot blot - pipet 1ul of the secondary antibody onto nitrocellulose, allow it to dry then develop the dot blot as before
B. add a postive control or Magic Mark to your gels next time around

Good luck
RB