3 replies to this topic

[VJbiofication]

Dear all,

We usually transfer SDS PAGE to PVDF membrane at 400mA for 60 min. I need to transfer only some amount of  protein from the gel to PVDF, so that I can do western blot on this membrane and use it as a guide to isolate spots from the still remaining protein in the 2D gel.

My question is how much of proteins would have transferred if I run the transfer for just 5 min or 10 min? Will I have some proteins in the PVDF membrane already? Or do I have to run may be just 2 min or longer like 15 min?

I need to have most of my proteins in the gel and only a few to be on the transferred PVDF membrane.

Thanks for any replies.
VJ.

[bob1]

You can do in-gel westerns where you detect in the gel you have run.  This will be a better option than trying to transfer a proportion of the protein onto a membrane.

Incidentally, the transfer would depend on the size (and partially on charge too) of the proteins.  Big proteins transfer slower than small.

[VJbiofication]

Thank you for your answer, but my question was about the optimal time for transfer of some amount of proteins to PVDF and leaving the remaining on gel. I can't do in-gel western(I need to take spots for MS analyses, will interfere) and I know that transfer depends on size of proteins.

[bob1]

There is no optimal time - this depends on the size and partially on the charge of the protein(s) you are interested in transferring.  It would also depend on the type of transfer you are doing, the voltage/amperage used, the size of the gel, and a number of other factors.