Big white spots on PVDF membrane - (May/13/2009 )

Hi! I'm using antibody from Santa Cruz, polyclonal antibody from goat to detect 16 and 18kDa protein. Initially, when I first did the western blotting, (after a few trials) I got good band. Then I prosessed new samples and the nightmares begin. Anyhow, it was only the unspecific bands, which can be disregard in my case.

The problem is that recently I am getting white big spots like blotches, seems like the transfer might not completed. I've incubated the membrane with GAPDH antibody which then showed a very good bands, means not the blotting problems. (Blotting, 90V 2 hours 12% gel, using wet blotting system, buffer MeoH 20% and SDS, membrane PVDF - incubate 1 minutes in MeOH).

So, it leaves me with a few other options:
1) uneven distribution of primary antibody
2) uneven distribution of secondary antibody
3) dropping Millipore detection solution on the membrane

Please help me by giving your opinons if my troubleshooting is wrong or you have some other comments.

Thank you.

Naba

I think your problem is how you're applying the detection solution. Try pipetting it onto the edge of the membrane, then tilt it to distruibute the solution over the entire membrane.