EMSA problems - (Nov/20/2006 )
I am new to this forum. Recently i tried to do EMSA for the first time and amzingly it worked the first time. I obseved a neat shift. My problem is I am unable to repeat the experiment since then. I am using lightshift EMSA kit from Pierce. I tried with re-extracted protein in case the original protein got degraded without much sucess. Why I am not able to repeat the experiment. I am following the same protocol (from Pierce) all the time. It is really frustating not to be able to repeat the experiment. Can any one of you with more experince with this assay please help me.
Did you get a good image? If so, just call it a "typical" expt, use it and move on...
What is exactly the problem you are having ? There can be many problems - too much protein can create non-specific interactions and aggregates which will not shift through the PAA gel. In that case you will see a dark spot at the beginning of the well and a weak smear in the lanes.
Maybe there is a problem with your dye ? What are you using for marking ?