EMSA to determine binding affinity of protein to
radiolabelled DNA
1.Add 9 ml of initial protein concentration to first tube. Add 6 ml EMSA buffer to 13 tubes. Remove 3 ml of protein from first tube and add to second. Mix and remove 3 ml from this tube and add to third tube. Continue these serial dilutions until there are 13 tubes with protein and one with only buffer.
NOTE: the actual protein concentration in the reaction will be 12% of the final concentration from this step. Adjust protein concentrations so that upper (20 nM) and lower (0.002 nM) baselines of binding are established.
2.Add 39 ml of EMSA buffer to each of the 14 tubes.
3. Heat DNA for 3 min @ 90¡C. Add 5 ml of 1000 counts of diluted DNA to each protein sample. Incubate reactions for 1 hr @ RT.
4. During incubation, pour 7% acrylamide gel in 0.5x TBE.
5. Pre run gel 30 min @ 300V.
6. Load 25 ml of each reaction onto pre run gel. In last lane, load bromophenol blue/xylene cyanol marker.
7. Run for 1 hour @ 300V or until bromophenol blue (~19mer) marker is 2/3 of the way down the gel.
8. Dry the gel under vaccuum at 80¡C for 1 hr.
9. Expose a phosphoimager plate for ~16 hr.
10 mM Tris-HCL 250 ml 2M stock
3mM MgCl2 150 ml 1M stock
0.1mM EDTA 20 ml 0.25 M stock
100 mMNaCl 1.25 ml 4 M stock
100mg/ml BSA 500 ml 10 mg/ml stock
0.02% IGEPAL 100 ml 10% stock
5% glycerol 2.5 ml or 3.14g
1 mM BME 1.06 ml/15 ml buffer
(can make stock and add BME directly before use, but stock only good ~2 weeks)