Transfer problem

[Anais1979]

Hi guys, I really need your help!

I have a transfer problem which I can't explain well.
This is what I do : after I assemble the sandwich, I do transfer in cold room, use ice pack, tank covered with ice, at 0.3 A for 2 hours. Or at 30V overnight.

Problem : there is no problem transfer for proteins of mv 80, 60, 50. All bands are even.

However, the protein of 25 mv seems to give me uneven density bands when I analyze the results. Seems like out of 8 lanes first 4 lanes are dark, and then the last 4 lanes are very light. This protein is "on top" of the tank.

I thought of these possible problems:

1) Protein is small, so can some of it "run off" the membrane, or would it all run off in this case. Also, why is it always 4 lanes in line where it vanishes?
And it can't be a loading issue because the proteins that are on the same membrane but with bigger weight are all even.

2) Uneven pressure? This protein seems to be near the place where the cassette closes. I also use 3 pads to increase pressure, so may be it is too much pressure on one side?

Any ideas? Will really appreciate your help!