Why do I have to almost "burn" my transfer before I can detect?? - (Oct/21/2009 )
This may not be a buffer or a membrane problem -- it sounds electrical to me. Have you tried a different power supply? Have you checked your transfer apparatus for broken wires or cracks? There's something wrong with the circuit if you're getting that much heat at low voltage -- what is the current? Something is causing too much resistance -- usually this is caused by an improperly made buffer, but you've tried several, so I'm begining to think it's an equipment issue... Do you cast your own gels?
But people transfer in cold rooms all the time, and the system is kept really cold. What's your take on that?
Feelcontraire on Nov 18 2009, 08:13 PM said:
medchemgirl on Oct 21 2009, 11:35 AM said:
I am completely puzzled by this. Every time I try to transfer a particular protein, it wont stick to the membrane unless I run it to the point where the buffer evaporates, almost like baking the system. I am using the semi-dry blotting system from Invitrogen. Even if I use the same amount of voltage (50V, Invitrogen protocol calls for 20V) and keep it really cold that it doesnt evaporates, I won't see my protein, unless I let it go until the buffer evaporates and it smells like burnt! I don't get it! is my protein that hard to to stick? its a 17KDa protein, very small!
I have done this multiple times as to determine that the only way I can detect it is by frying the membrane!
Any comments please?? I'm about to call Invitrogen and ask about this.
Well,this is because heat impairs the gel structure and allows proteins to flow through smoothly. To achieve even transfer temperature should be over 65ºC
Well, no, in a semi-dry system 60V is a lot and u will evaporate the little amount of buffer u have in an hour. My gels are precast Bis-Tris 4-12% from Invitrogen. I did tried the transfer in the regular tank system with no results. I went back to the semi-dry and doing the transfer 30min at 20V and now I'm seeing ghost bands!!!!!!! but not the ones where u see the center of ur band in blank, only if I over expose I can see my band in "white". I'm just loading 10ug!!! how can this be?
HomeBrew on Nov 19 2009, 11:11 PM said:
Here's a silly question: what happens when you transfer for 20 minutes? (Otherwise, I have no suggestions.)
Do you have a reason why?, I could try but I don't see what would change from 30 to 20 mins.
lab rat on Dec 2 2009, 08:17 PM said:
Maybe your protein is so small that it blows through the membrane if transferred for too long. But I re-read the earlier posts, and saw that you were using 0.22 um membrane now.
Yes I am. First was using nitrocellulose and now Immobilon Psq, I checked and both are 0.22um.
lab rat on Dec 3 2009, 01:02 PM said: