Problems with non-radioactive EMSA signal - (Sep/27/2007 )

I am currently trying EMSA with the pierce lightshift EMSA kit. The signal I got sometimes is so weak that only my positive control shows up even though I had expose the film to the membrane for 3-5h or even overnight. Because the nuclear extract from my primary culture cells produce weaker signal, this problem makes my results not useful at all.

Does anybody know how to get stronger signal? what could be the reason causing the problem? Do I need to crosslink the membrane longer than usual? I have followed the protocol very well but I got big variation from one to the next experiment. I have also prerun the gel to allow the mA stay constant---to get rid of extra APS. Is it possible to reprobe the membrane? I tried once but got no signal at all!

Help, Help! So frustrated!!

Thanks!!!

you may need more antibody? or, better antibody?