- 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
- To some chromatin suspension add concentrated urea and concentrated
NaCl separately to yield a final concentration of 7 M urea and 3 M NaCl.
- Centrifuge the clear solution at 85,500 xg for 48 hours at 4°C to pelletextracted
- 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.
- 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.
- 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.
- Apply 20 µL samples of the redissolved protein extract to 0.6 × 8.0 cm
polyacrylamide prepared as in step 4.
- The gels are run against 0.9 M acetic acid in both upper and lower baths
for approximately 3 hours at 100 V.
- Stain the gels for 1 hour in Coomassie Blue, rinse with water, destain, and
store in 7% acetic acid.
- 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.