Transfer problem - why the size of SDS-PAGE become too small during the transfer (Oct/17/2007 )

Hi dear All,
My western experiment troubled me for a long time as the transfer step did not work(TANK TRANSFER SYSTEM).

The proteim size >60kDa can not be transfered to the nitrocellulose membrane, but the transfer worked well a month ago.

Each time I saw the same problem, the size of my SDS-PAGE become small. As the size of the filter paper and the membrane is bigger than the gel, the edge of the top and bottom filter paper and the membrane contacted each other. The protein>60KDa always showed poor transfer efficiency.

My question is why the gel became too small? The gel was runned good as the prestained marker looked normal during the running process. Are there any problem with reagents for the SDS gel, such as polyacrylamide ? Tris.cl?
Does anybody have this kind experience? Thanks in advance!

You mean this is a new problem you didnt face before so did you change anything in your steps?

about gel getting smaller in size did you soak your gel enough time in transfer buffer before blotting as for 15 minutes . i do this because gels put in methanol will shrink over time and we don't want the size of the gel to shrink during the blot, so soak it longer if the gel is continuing to shrink.
hope this help

I'm not sure the problem is soaking time.
I don't soak the gel, because I prefer to transfer the gel to the membrane directly from the glass plate (no bubbles) and I never had my gel shrink.
Do you use a new batch of buffers?
Check your buffers.

If you have a high amount of methanol in your gel, they can shrink, as spanishflower said. I once put my gel directly in methanol (instead of the membrane!) and it was very very small afterwards, did not look healthy So, perhaps you can reduce the amount of methanol (15% should be enough) or you put them in transferbuffer for longer time, as mentioned before. I prefer semi dry blot, have never seen this effect in there.

hi missele

well this is the first time for me to know that we dont have to soak the gel i had been taught that after running an SDS/PAGE gel, immediately equilibrate the gel in a small container of transfer buffer for 15minto help facilitate the removal of electrophoresis buffer salts and detergents. If the salts are not removed, they will increase the conductivity of the transfer buffer and the amount of heat generated during the transfer. Also, gels lower than12% acrylamide will shrink in methanol-containing buffers. Equilibration allows the gel to adjust to its final size prior to transfer.

Dear All,
Thank you for all of your nice reply.

I have done western at least 6 years since I started my Ph.D., I never soak the gel in transfer buffer and my western worked fine.

I listerned a western seminar presented by Millipore company in this July. From their seminar, I knew if the gel was banlanced with the transfer buffer, it will not change the size obviouly. SO I soaked the gel for 5-10min each time. and the western works well in the first several times. But recently, the western did not work as the protein>60kD never transfered to the membrane and the gel became too smaller. I changed a new batch transfer buffer, and still have the same problem.

I guess I should also change the acrylamide/bisacrylamide as it has been stayed in my 4C refrigerator for 2 years.

Thanks!

I have never add methanol to my transfer buffer and my western work fine