Hi Sonali,

Can I suggest you to try a 15% resolving gel?

For transfer, maybe you can try holding the voltage at 100V for 1 hr?

This is what we do in our lab. Hopefully this method will work for you.

Mel

Smaller proteins can definitely pass through the membrane more easily than larger ones. So, three things come to mind. 1) Adjust your level of MeOH from 10% - 20%; this may help. 2) Decrease your voltage or time of transfer. I usually use 1 hr at 115 V in the cold room, you could go lower as Mel suggested. 3) Place a second membrane behind your first to "catch" any blow through. Good luck!

Hi

I am using 20% methanol in my transfer buffer and transfering at 80V for 1hr. still its giving me trouble.

Hi Sonali,

Do you have any picture of the blot to show us? Perhaps with the help of a picture we might be able to come out with some solutions.

Mel

Hi Sonali!

I run the transfer for 17KD protein at 70V for 40-45 mins. Although with 0.2uM PVDF membrane the chances of protein passing through is unlikely. But reducing the time of transfer might help.

Best

Hello everyone,

Thank you so much for your valuable time and inputs. Finally I could find a way out. For past few couple of transfers it was worked well. Following are the changes I made in my protocol.

1. I used 6 filter papers instead of 2 to make my blot sandwich. Probably this enhanced the contact between the gel and membrane.

2. Transfer condition : 80V for 2h

This has worked for me in my lab.

Regards

Sonali