EMSA problem

[Balat]

I am new to this forum. I have a problem with EMSA. Two weeks ago when i tried emsa for the first time amzingly it worked and got a shift. I tried to repeat the experiment many times without success sofar. I have tried using newly extracted nuclear protein as I was freeze-thawing my protien many times but no shift observed. How critical is to keep the binding reaction on ice before icubating at RT as i am doing all my binding reactions at RT and incubating the reaction at RT for 20 mins. I am usnig light shift chemiluminiscent EMSA kit from Pierce. Please help

[moleculardude]

Hey there Balat!, welcome to the forum.

For some proteins it is pretty critical to keep things on ice as your protein may be degraded quickly in its active form, or it could be dephosphorylated preventing binding to DNA.

What I would do is keep things on ice. Doesnt hurt. I would not freeze thaw, aliquot your samples and make sure you have protease inhibitors.

Keep everything on ice. Once you master the technique, then you can be more sloppy like me I actually incubate at 37C lol...

Please post your results here.