Can ice-pack be a problem?

[Anais1979]

Usually I was transfering 2 gels at 300mA for 2.5 hours in cold room with good results using the Bio-rad mini transfer system, and I would put ice pack in the system at the beginning of the process.

Our lab tech told me, that I need to change ice pack after one hour and that would increase the quality of transfer since proteins transfer better at lower temp.

So I did. And something pretty weird happened -
By the way, the ice pack was unfrozen with ice floating when I got the system out:

First of all, the top markers were still on the gel - on both of them, but most of the ladder transfered equally to both membranes.

But then I soaked membranes in ponceau, and I found out that one membrane transfered ok (but not as good as usual), but the other one has almost no protein on it!

It is the same gels, and same samples that I am using. And the equipment is working fine - I checked it today with the technician.

So I came to the conclusion, that the only thing that could cause it is actually that second ice pack...

Do you guys have any ideas if the ice-pack can do that trick, or I need to think what else could make that membrane protein-free? May be too cold is not that good either...

Thanks!

[Anais1979]

Never mind, bad transfer has nothing to do with the ice-pack.

[quest]

I usually do change the ice block like as you had mentioned but my gels are always fine. Did you check the orientation of the second gel which had no proteins on it! Sometimes it happens by mistake. Anyways, the ice-pack is not at all a reason.

[Warthaug]

Are you using a sealed ice-pack (i.e. the "picnick ice packs"; i.e. the blue gel) or the open canister systems? If you're using the later the melted ice can, on occasion, end up in the transfer buffer, diluting it. That can screw up your transfer, although it doesn't sound like it in this case.

I usually put the transfer apparatus in an ice bath, and put the ice pack in the transfer container itself. This way I don't need a cold room, and can keep the system very cold simply by replacing the ice as it melts.

Bryan