2 replies to this topic

[qpwoei4756]

Hi everyone,
I am planning to perform EMSA with a supposedly DNA binding protein and a 22-mer oligo DNA. But I was wondering what's the difference between using purified protein fraction versus cell extract extracts + noncompetitve DNA such as dIdC?
My protein is transiently transfected to human cells using lipofectamine 2000. I can already have my protein of interest purified from 293T human cells using anti-FLAG beads, but I can only get this much (see attached picture) protein. It's also time consuming and costly. I already tried to use the purified protein to do EMSA several times and I get little to no binding.
I'm reluctant to use the cell lysate because the expression may not be enough. Is this much protein expression enough for whole cell EMSA?

Thanks
qp

Attached Thumbnails

• 

Edited by qpwoei4756, 08 February 2013 - 05:44 PM.

[pcrman]

whole cell lysate can give you a lot of non-specific binding. I think you can try expressing your protein in bacteria and use the purified protein for EMSA or use nuclear extract from HeLa cells. For proteins to bind DNA, it must be nuclear so nuclear extract is typically used for EMSA.

[qpwoei4756]

pcrman, on 08 February 2013 - 06:02 PM, said:

whole cell lysate can give you a lot of non-specific binding. I think you can try expressing your protein in bacteria and use the purified protein for EMSA or use nuclear extract from HeLa cells. For proteins to bind DNA, it must be nuclear so nuclear extract is typically used for EMSA.

Thanks for your reply. I purified a huge amount of recombinant protein expressed in E coli and tried it on my DNA, but there were very weak or no binding. However, I think that proteins purified from transfected human cells like Hela and 293T cells may have post-translational modifications that bacteria can't provide. Both the protein and DNA are from human by the way.
I was wondering if the DNA-protein binding can be improved by using the whole cell lysate of my human cells instead of purified proteins. Of course, I will include a negative control mutant DNA that's not supposed to bind to my protein of interest.