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Electrophoretic Mobility Shift Assays (EMSA)

Reagents:

Binding Buffer : Make fresh or aliquot and store frozen

20 mM HEPES pH 7.9
50 mM KCl
1 mM DTT
0.1 mM EDTA
5% glycerol
200 m g/ml BSA

nonspecific DNA:

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 g d(I:C) and 0.5 g ssDNA.

Nuclear Extracts:

Prepared according to our modification of the Dignam procedure. Diluted to 1 mg/ml with 1X binding buffer just prior to use.

Gel:

4-6% non-denaturing polyacrylamide

For 45 ml of 4%, 1.5mm thick gel
6 ml 30% acrylamide (29:1 acrylamide:bis ratio)
4.5 ml 5X TBE
34.5 ml H2O
300 l 10% ammonium persulfate
30 l TEMED

Running buffer:
0.25X TBE

Probe:
0.5 ng 32P-labeled dsDNA (typically 50,000 cpms) in 1 l

EMSA Reaction:

Mix all components except nuclear extract and labeled probe: Add the requisite amount of extract (2.5-5l) 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 l 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 l 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.

 

           

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