Non-radioactive labelling for EMSA? - (Oct/10/2005 )
I am wondering if it is possible to label the DNA with non-radioactive tag for EMSA. If is possible, can I have the protocol?
I know one method using DIG to label DNA probe, after gel running, transfer DNA/protein on to membrane and use anti-dig antibody for detection. This method is safe, but tedious.
alternatively, a much simpler way is to use some kits, like EMSA kit from invitrogen with sybr green and sypro ruby. But I don't know its sensitivity.
DIG EMSA protocol
works pretty well (results in press, with shifts and supershifts; I can send you a pic if you want proof)
let me know if you decide to use it and you have questions
oh, hey, I need to add something
if you choose to label your oligos with biotin, they have to be end-labelled or you might interfere with binding to the transcription factor (the biotin may contribute to some inhibitory secondary structure); that is why I used Pierce's 3' end-label kit; a year ago when I developed the method there were no other DNA biotinylation kits on the market that would only add the biotin label to one end of the oligo; there may be others now
I have used it to confirm and analyze AP-1 and NF-kB binding to a couple eukaryotic promoters