Analysis of DNA-Binding Activity in Neuronal Tissue with the Electrophoretic Mobility-Shift Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 315

13. Proceed to EMSA or quickly freeze aliquots in liquid nitrogen and store
at -80°C.

3.4. Preparation of the Gel
A nondenaturing, 4% acrylamide- bis -acrylamide gel (37.5 1) is used with
vertical electrophoresis for EMSA. TBE buffer is used as running buffer at
0.25X.

1. Assemble the glass plates and spacers. Both plates and spacers need to be washed
with water and rinsed with ethanol. The plates need to be spotless, but soaps
should be avoided because they may interact with the gel matrix during the run
and degrade the gel. We use 1.5-mm spacers for EMSAs.

2. Mix for a 30-mL solution: 0.75 mL of 10X TBE, 1 mL of 100% glycerol, 4 mL
of 30% acrylamide- bis -acrylamide solution (37.5 1), and 24 mL of distilled

water. Mix well and make sure glycerol is entirely dissolved.

3. To start polymerization, add 300 µ L of 10% APS and 30 µ L of N , N , N , N -

tetramethylethylenediamine (TEMED).

4. Quickly pour solution between glass plates, insert comb, and let rest for at least
30 min to polymerize.

5. Prerun gel in the cold room (4°C) for 30 min at 200 V with 0.25X TBE.

3.5. EMSA

1. To make 2.5X EMSA buffer, mix on ice 50 µ L of 5X EMSA buffer, 45.25 µ L of

distilled water, 2.5 µ L of poly (dI-dC), 1.25 µ L of MgCl 2 (stock: 1 M ), 0.5 µ L of

DTT (stock: 1 M ), and 0.5 µ L of PMSF (stock: 100 m M ) ( see Note 7 ).

2. Take 6 µ L of 2.5X EMSA buffer/sample.

3. Add 4 µ L of protein solution (or 4 µ L of sonication buffer for the control lane

without protein).

4. Incubate 10 min on wet ice for nonspecifi c binding to poly (dI-dC).

5. Make a stock solution of 4 µ L of distilled water and 1 µ L of labeled oligonucle-

otide for n + 1 samples.

6. Add 5 L of water and labeled oligonucleotide stock solution to each sample

(= 1 ng labeled oligonucleotide per sample).

µ

7. Mix by pipetting.

8. Incubate at room temperature for 10 min for protein-DNA binding.

L onto a prerun gel in the cold room, and start with control sample
without protein. Flush out the wells of the gel with running buffer before loading
the samples.
10. Load dye in adjacent wells. Do not load dye with sample because it interferes
with the electrophoresis ( see Note 8 ).
11. Electrophorese for 1 h to 90 min at 200 V in a cold room. Bromophenol blue
should migrate two thirds through the gel. In a 4% gel, the bromophenol blue
migrates at approx 40 bases. Figure 2 shows some examples of EMSAs.

9. Load 10 µ