EMSA -- Chemiluminescent version by Pierce - (Aug/22/2006 )
Hi All. I have just started using Pierce's chemiluminescent EMSA Kit, and I am having some trouble. I was hoping that some of you with experience with the kit might be able to help me with some optimization.
I have been running an 8cm x 10cm x 0.75 cm 4% acrylamide gel.
What percentage gel do you find works best, or does it depend on the probe and nuclear extract of interest?
At what voltage do you run your gels, and at what temperature?
At what voltage/amps do you run your transfer, and at what temperature? Pierce recommends that the 0.5X TBE be cooled to 10o in a circulating waterbath. Instead, I have been running my transfer at 4o without pre-cooling the TBE. Perhaps I should do otherwise....
Are there any other "tips," "tricks," or variations from the protocol that you would recommend?
Thank you so much for any/all advice that you can give me!!
I am not using Pierce's Kit but am doing a radioactive EMSA. But I think some principles are the same, like the gel. I am using a 6% Polyacrylamide Gel (19:1). The percentage you use depends on the size of the protein-DNA-complex, you want to see. Normally, you can use something around 4-7% and look, what works best for your samples.
I prerun my gel at 150V for 30 minutes and run at 200 V for 2h, all at room temperature, because I have to work in the isotpe lab without a cooling possibility. But I tried once a run on ice, looked the same as at room temperature. I just cool my buffer prior to run.
For the buffer I use 0,25xTBE.
I fix my gel after run and dry it at 80°C for 1,5 h. Then the autoradiography at -80°C over night.
Hope, I could help a little.