High Molecular weight protein separation - (Oct/29/2009 )

Hi everyone!

I got a problem in separation of two high MW proteins after a co-IP. One of them is 220-230kDa the other one is ~190kDa. The problem arises because the smaller protein "diffuses" on 6% gel when I run the IP sample for a long time. This results in a black smear in the lane when I detect with the antibody against the larger protein on western. I guess there is so much of protein pulled-down that it is being recognized non-specifically by the antibody.
So how can I achieve a proper separation of the two peoteins, but avoid non-specific band recognition? Shoul run a 4% gel in the cold?
or use gradient gel? or swithch from Tris-glycine system to something else?

Well, please feel free to give any kind of advice - any input is greatly appreciated!

Happy Halloween by the way!

Dimilletronc

Try a 4-8% gradient, it should work fine.

bob1 on Oct 30 2009, 05:47 AM said:

i agree and also try not to overload as overloading will increase diffusion.. also at this high a pore size the protein will anyways be a bit diffused and wont give sharp bands...
I think you can work it out in this buffer system itself!!!
Best luck!!