hi Remi Joe,

Mainly, it could be because of Power (Voltage or current) issues which affected the transfer.

However, if it's very high MW protein and not resolved in respective suitable %age of gel then something such smearing can be seen.

Well, if you provide little more basic details such as MW of protein, Gel %, Transfer voltage, transfer buffer composition, and pH, etc. , then members can give more satisfactory answers.

Regards

Saurabh

Saurabh gave a good answer for the first steps, the voltage/current could be the issue or the gel is too tight for the proteins in question

What is that split on the rhs of the imagel and when did it happen. If the gel split during electrophoresis then the current through the gel will not be uniform which may account for the strange movement

A little extra detail would help, but let's take out the crystal ball.

• Are you using tank or semi-dry transfer? Tank blotters cause less heat stress and hence give better blots.
• I found 30-40 V for 1 h good conditions for transfer, higher voltages increase the heat problem and who knows what happens during longer times
• I use Dunn's transfer buffer instead of Towbin's (10 mM NaHCO3, 3 mM Na2CO3, doi:10.1016/0003-2697(86)90207-1). For the large transmembrane proteins I'm interested in I add 50 µM SDS
• Of course, you need to avoid air and steam bubbles (which might have been the cause of smearing of the middle blue bands)
• If your transfer apparatus contains cooling loops or ice containers, use them

Engelbert Buxbaum

I do a wet transfer at 30V for 1 hour 30 minutes in cool conditions. I made sure to roll out any bubbles but could you elaborate on steam bubbles?

Especially in semi-dry blotters there is a lot of heat in a small volume. This leads to bubbles for two reasons:

Hi Remi Joe, I think this happened because the gel or the membrane moved away from each other during transfer, mainly because the transfer sandwich was not securely fixed in the chamber. You can try to add one more or two whatman paper and see if it fix the problem.

Good luck :)

Are you sure that smearing happens when you transfer? To me it looks as if there is some problem with gel itself. Did you try to stain it to check whether this idea is correct?

Remi Joe

It is because of the gel making and the power fluctuations. You need to optimize on that portion of the protocol !!!!