This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Chapter 10: Chromosomes

Exercise 10.4 - Chromatin Electrophoresis




  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.

Return to Table of Contents

Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --