Problem with radioactive EMSA, help! - (Jul/26/2005 )
Hi everybody,
I am currently trying to purifying a novel transcription factor from crude protein extract. The only thing I know is the DNA sequence that the protein binds to.
My problem is that, everytime after I have done a purification step, either cation exchange or gel filtration, I found the protein sample running on a EMSA gel getting weird.
The treated protein sample has already been dialysed against a lower salt buffer. However, after I run it into a EMSA, there is serious smearing effect. Also, a lot of stuffs seem to be stucking in the well rather than run into the gel.
I don't know what can I do, may some smart guys please help me out. Thanks! 
Have you thought about an affinity purification using bound DNA with the correct binding sequence?
You can order oligos with an acrydite modification (IDT, among others) which allows you to copolymerize the oligos in a PAGE gel. You could make a single oligo which hairpins to form a ds recognition site for your protein.
You can order oligos with an acrydite modification (IDT, among others) which allows you to copolymerize the oligos in a PAGE gel. You could make a single oligo which hairpins to form a ds recognition site for your protein.
I may try that, but at the time being, I need to solve the problem of EMSA first. Do you have any suggestion? May stuff is being stucked in the well but not running into the gel. help!