I have never seen anything like this! I don't think its air bubbles between the gel and the membrane - they usually give smooth-edged white areas. But how many times have you seen this result?

To eliminate a problem with the electrophoresis step, have you tried staining a gel with coomassie after the run? Or even staining the "residual" gel after electroblotting?

Try running  a simple commassie gel with your samples and see the pattern of your samples. If you observe clear bands at your expected molecular weight, then everything is fine with your samples. Next run a gel and transfer it at 30V in a cold cabinet after adding methanol to the transfer buffer. Alternatively you can run it at a higher voltage for 2 hours but maintain the cold condition. If you have a semi dry transfer, transfer at 60V for 50minutes RT. Then do a ponceau Staining to see if everything went well with your transfer. Accordingly you can proceed with your western. Cheers!

You have some problem of contact between gel and membrane. You need to change the sponge or add one more to increase the pressure.

Hi Piotr,

could it be, that your membrane is somehow "contaminated"? It looks if something is one the membrane.

Best regards,

Carsten

I'm assuming you have rollered the gel during set-up? If there is air in between the gel and membrane, it doesn't matter how much pressure you put on it, the air will be trapped in the layers. :)

As the lack of an appropriate contact between gel and membrane is unpredictible, I don't think we should asume bubbles have to be nice circles. For me, they look like bubbles. Keep us posted.