Electrophoretic Mobility Shift Assays (EMSA)
Binding Buffer : Make fresh or aliquot and store frozen
20 mM HEPES pH 7.9
We typically use poly d(I:C) (Boerhinger Mannheim #108812) and/or sonicated salmon sperm DNA. Often the proper concentration and combination of DNA needs to be determined empirically and depends somewhat on the probe and source of nuclear extract being used. A good starting point might be 1 µg d(I:C) and 0.5 µg ssDNA.
Prepared according to our modification of the Dignam procedure. Diluted to 1 mg/ml with 1X binding buffer just prior to use.
4-6% non-denaturing polyacrylamide
For 45 ml of 4%, 1.5mm thick gel
Mix all components except nuclear extract and labeled probe: Add the requisite amount of extract (2.5-5µl) and incubate at 15o for 15 minutes. This allows nonspecific protein:DNA interactions to occur or for the specific competitor to interact with the factor of interest. Add 1 µl of the labeled probe and continue incubation at 15o for an additional 15 minutes.
Load the reaction onto the gel while it is running at about 25V. Load a few µl of gel loading buffer containing dyes to an unused lane or to the probe alone control as a marker. Run gel at 150 V until bromophenol blue is about 2-3 cm from the bottom.
Dry the gel onto blotting paper at 80o for approximately 45 minutes and autoradiograph.