Exercise 10.4 - Chromatin Electrophoresis

LEVEL II

Materials

• 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 -mercaptoethanol
  
• 0.25% Coomasie blue

Procedure

1. To the chromatin suspension from Exercise 10.3, 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 pellet extracted DNA.

3. Collect the supernatant and dialyze it against 0.05 M acetic acid (three 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 (Exercise 4.1), 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 -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 x 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 Coomasie blue, rinse with water, destain and store in 7% acetic acid.

9. If densitometry measurements are made, 5 µg of pea bud fraction IIa protein has a density of 1.360 density units x mm with a 95% confidence limit of 10%. By comparison, the density value can be used to quantitate the concentration of protein fractions in µg of your sample.

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