problem with transfer during western blot - western blot /sds page (Jul/01/2008 )

hi
I am doing western blot and i am running 40ug of protein and run 100V for about 1.30 mins , i transfer in cold room at 100V for 2 hrs. It worked fine for the first time but something is wrong with my transfer this time .
my transfer buffer is 100ml of transfer buffer 1X, 200 ml methanol and 700 water. i soak the gel and blot paper (PVDF) and whatnam ppaer from 5-10mins before asseble the wet transfer. When i check tranfer with ponceau i can see the top band very faint but not the small one, my protein is tranced protein so it ranges form 75 to 16 kd (each sample has different size) and i need to run in the same gel please help.
Ps my gel is 12%

-mama wa nyumbani-

Hi

It all sound ok. Do u use the same membrane (pore size)?

U can also had SDS 2.5% or more to the transfer buffer.

The transfer buffer spoused to be X10 and not X1 as u wrote (am i correct?). cool the transfer buffer on ice

before using, it helps.

try to load more protein on gel

good luck.

-amtash-

QUOTE (amtash @ Jul 3 2008, 06:05 AM)

U can also had SDS 2.5% or more to the transfer buffer.

you should not use more than 0.05% sds in the transfer buffer. too much sds will interfere.

-mdfenko-