Bloting IEF gels onto PVDF membranes? - (Jul/24/2007 )
I’m having a bit of trouble transferring my IEF gels. I’m trying to blot for p53 (after the IEF gel run) and I use 0.7% acetic acid to transfer in the reverse orientation. I took this to mean that I have to swap around the membrane and gel so the membrane is on the cathode side of the gel. I transferred for 10V overnight. The following day I stained the IEF gel with comassie to see if the transfer worked and there was protein still on there, also nothing showed up on the membrane after development.
I cant figure out what I did wrong , any help would be much appreciated.
P.S sorry if Im rehashing a done topic .
a couple of questions:
(i'm sorry if this one appears insulting) is your ief gel on an acetate support? this will block the current from the gel.
did you equilibrate the gel in transfer buffer before attempting to transfer? your proteins should have a net neutral charge after focussing. you will need to remove ampholytes and "recharge" your proteins before they will migrate. this can be done with towbin buffer and normal placement of electrodes as long as your proteins of interest have pIs below the pH of the buffer.