Accidentally transferred reverse! Help me! - (Jan/26/2006 )
I did SDS-PAGE, then transferred onto PVDF, but accidentally reversed the anode and cathode plugs for the first two minutes of transfer. Then I realized the mistake and corrected it. Do you think all the protein will have escaped and lost in those first two minutes?
Actually I am typing this question as I am still transferring (in the correct orientation)...
So I don't know the answer, but I'd really appreciate some thoughts just to allay my concerns...
I speculate that some protein loss is of course inevitable. However, I think maybe 90% will still have been in the gel, and hence reversing the current would have allowed the proteins remaining in the gel to be transferred to the membrane...
2 minutes !
don't worry, it's fine !
unless we would never transfer for one hour !
It happened once I forgot to put the membrane, I realize after 15 minutes. results was still fine.
(of course it depends on the quantity of antigen)
you can smile, it will work
Laurence, thanks for the reply.
The result was as you said: the proteins transferred properly to the membrane. I had been imagining that proteins transfer fast at the outset of transfer, and that the reason why you transfer for over an hour is so that the high molecular weight proteins that are slow to transfer can transfer completely...
Anyway, this was the semi-dry transfer. I usually do 80mA per minigel membrane for 80minutes. Which is better anyway, the semi-dry transfer or tank? I heard that it is easier to get bubbles when you do the tank transfer.
I don't know which one is better.
I use one or the other, depending on the material I find in the lab