Chromatin Electrophoresis

• 14 M Urea
• 6 M NaCl
• 0.05 M and 0.9 M acetic acid
• Dialysis tubing
• Electrophoresis apparatus
• Prepared gels
• 10 M urea-0.9 N acetic acid-0.5 M β-mercaptoethanol
• 0.25% Coomassie Blue

1. To some chromatin suspension add concentrated urea and concentrated NaCl separately to yield a final concentration of 7 M urea and 3 M NaCl.
2. Centrifuge the clear solution at 85,500 xg for 48 hours at 4°C to pelletextracted DNA.
3. Collect the supernatant and dialyze it against 0.05 M acetic acid (3 changes, 6 liters each at 4°C). Remove the dialyzed protein solution and lyophilize it to dryness.
4. Meanwhile, set up a standard polyacrylamide gel, using 15% acrylamide (15%T:5%C) in 2.5 M urea and 0.9 M acetic acid. Set up the gel in the electrophoresis unit and run the gel at 2 mA/gel for 2 hours with no sample, using 0.9 M acetic acid for the running buffer.
5. Dissolve the lyophilized protein from step 3 in 10 M urea-0.9 N acetic acid-0.5 M β--mercaptoethanol (to a final concentration of 500 micrograms protein per 100 µL of buffer) and incubate at room temperature for 12–14 hours prior to the next step.
6. Apply 20 µL samples of the redissolved protein extract to 0.6 × 8.0 cm polyacrylamide prepared as in step 4.
7. The gels are run against 0.9 M acetic acid in both upper and lower baths for approximately 3 hours at 100 V.
8. Stain the gels for 1 hour in Coomassie Blue, rinse with water, destain, and store in 7% acetic acid.
9. If densitometry measurements are made, 5 µg of pea bud fraction II, a protein, has a density of 1.360 density units × mm with a 95% confidence limit of 10%. By comparison, the density value can be used to quantitate the concentration of protein fractions in mg of your sample.