Here's another picture of another membrane stained with ponceau S post-transfer. All my membranes are looking like this now..

Nomi

Very first reason that you have to think of is the dry area of membrane where your protein is not binding. Is it nitrocellulose membrane ? If it is then you may boil water at 55C or higher and immerse your membrane in it and later move it to equilibrate in the transfer buffer for considerably good time. Whatever your membrane is please make sure that your membrane is equilibrated.

Minor possibilities :

Before setting assembly - Did you wash extensively so that your residual acrylamide is washed off from the gel. Content of transfer buffer - Does it have SDS. If you can borrow some transfer pad from other lab which are relatively new you can check if it is problem of assembly itself.

Hi Mahesh,

Thanks for your answer. We equilibrate the nitrocellulose membrane in transfer buffer for about a minute before assembling the sandwich. We do not rinse the gel in transfer buffer before putting it into the transfer assembly (per manufacturer's instructions for our gel apparatus) - could this be an issue? There is no SDS in the transfer buffer. We have borrowed the sponges from a different lab as well to see if this is the problem, but no luck.

Hi Nomi, Equilibrate everything in the same transfer buffer- Try like above. I wash gel with water for 5 min and then with the transfer buffer for 15 min. Equilibrate your membrane, pads, and papers for 30 min and see. As of now, it dosent look like sponges is your problem.

Hi,

Im facing now the exact problem of yours.

Did you find the solution?

Thanks

We never found the definite cause...but we changed a few things and it's basically solved (so far so good :).

1) We changed the sponges in the transfer apparatus (now we use regular Scotchbrite pads cut to the right size) and we switch them out if they start to get thin.

2) I rinse the gel in transfer buffer for a few seconds before assembling the sandwich to remove traces of sds from the running buffer and to cool the gel off.

3) I soak the sponges in transfer buffer the whole time the gel is running (about an hour usually), and before that I rinse them briefly in water.

4) The nitrocellulose membrane I soak for 2-3 minutes in transfer buffer before putting it in the sandwich.

We also changed to a fresh ponceau staining solution, fresh reagents for preparing the running/transfer buffers, and fresh TEMED for the gel. Good luck!!