GAPDH not straight - (Oct/14/2008 )

Hi everyone,

I'm new to the lab and have been running western blots for about 2-3months now. Lately when I'm doing my GAPDH loading control, some of my bands are not straight. A few (like 2 or 3) bands look wavy or kind of swirled. Any ideas why??

Thanks

-Melistasy-

Do you use precast gels or do you cast your own?

There may be some irregularities in your gel that make the lanes uneven.

-Madrius-

If it is not irregularities in the gel, then it could be "north-western" effect. Basically the little bit of buffer between the gel and membrane in the transfer mixes a bit causing a swirl, usually due to heat in the transfer process.

-bob1-

Or maybe your gels are "smiling" because you are running your electropheresis a bit too fast and the gel gets warmed up? But this would also be visible in your other protein bands...

-biomaus-

I use precast gels from Invitrogen. Usually the other bands are fine, its just the GAPDH bands that are affected. How can I prevent the "north-western" effect??? I use the iBlot system to transfer.

Thanks everyone

-Melistasy-

QUOTE (melistasy)

I use the iBlot system to transfer.

This may be part of the problem, the iBlot produces huge amounts of heat during transfer, and the packs go off if stored for too long (approx 6 months IIRC).

The north-western effect shouldn't be a problem for dry transfers like the iBlot, usually it is only a problem in wet transfers.

-bob1-