- Bovine or porcine brain
- 0.25 M sucrose containing 0.0033 M calcium acetate
- 2.0 M sucrose with 0.0033 M calcium acetate
- 0.075 M NaCl with 0.024 M EDTA, pH 8.0
- Tris-HCl buffer, pH 8.0 with the following molarities, 0.05 M, 0.002 M,
- TCA (Trichloroacetic acid)
- Tissue homogenizer
- Refrigerated preparative centrifuge
- Bradford or Lowry protein assay
- UV spectrophotometer (optional)
Homogenize approximately 30 gms of bovine or porcine cerebellar tissue in a
teflon-glass homogenizer in 9 volumes of cold 0.25 M sucrose containing 0.0033
M calcium acetate.
- Filter the resulting brei through several layers of cheesecloth and obtain
crude nuclear pellets by centrifuging at 3500 xg for 20 minutes.
- Resuspend the nuclear pellet in 80 mL of cold 0.25 sucrose containing
0.0033 M calcium acetate.
- Obtain 3 cellulose nitrate centrifuge tubes and place 25-mL, aliquots of the
resuspended nuclear pellet in each. Carefully pipette 5.0 mL of 2.0 M
sucrose-0.0033 M calcium acetate into the bottom of each tube. Insert a
pipette with the 2.0 M sucrose through the suspended nuclei and allow
the viscous sucrose to layer on the bottom of the tube. Centrifuge the tubes
at 40000 xg for 60 minutes.
- Use the resulting nuclear pellet just above the dense sucrose layer. It is
used to extract chromatin proteins. Carefully remove the supernatant above
the pellet with a pipette. Then, insert the pipette through the nuclear layer
and remove the bottom sucrose layer. The nuclear pellet will remain in the
tube. Resuspend the pellet in 40 mL of 0.075 M NaCl-0.024 M EDTA,
pH 8.0 and centrifuge at 7700 xg for 15 minutes.
- Remove and discard the supernatant, resuspend the pellet once again in
40 mL of 0.075 M NaCl-0.024 M EDTA, pH 8.0 and centrifuge again at
7700 xg for 15 minutes. Repeat this process one more time.
- Resuspend the nuclear pellet in 40 mL of 0.05 M Tris-HCl, pH 8.0 and
centrifuge at 7700 xg for 10 minutes.
- Repeat step 7 to thoroughly wash the nuclei and then wash twice each in
0.01 M Tris pH 8.0, 0.002 M Tris pH 8.0, and 0.0004 M Tris pH 8.0.
- Resuspend the final washed nuclear pellet in ice-cold distilled water to a
final volume of 100 mL and allow to swell overnight at 4°C. Gently stir
the mixture on the following day. This solution is the pure chromatin to
be used for subsequent analysis.
- Determine the purity of the chromatin sample within the nuclear pellet
using one of the following:
- Determine the protein concentration by Lowry or Bradford procedures.
- Measure the optical absorbance at 230 nm (UV). The absorbance of a
1 mg/mL concentration of pea bud histone at 230 nm equals 3.5 OD
units. The absorbance follows the Beer-Lambert law, and is linear with
histone concentration. Since it is merely an optical reading, the sample
is not destroyed in the measurement.
- Measure the turbidity of the solution. Add trichloroacetic acid to a final
concentration of 1.1 M and wait exactly 15 minutes. Read the OD400. A
10 µg/mL solution of pea bud histone has an OD = 0.083 at 400 nm. This
technique is excellent for readings between 0 and 0.15 OD. The
TCA precipitates some proteins and, thus, this procedure is more
specific to histones than B. It can also be performed without a UV
- Measure by nondestructive fluorometry. Histones can be detected by an
excitation wavelength of 280 nm and a fluorescence measurement at
308 nm. Nonhistones can be detected in the same sample by excitation
at 290 nm and measurement at 345 nm. Of course, this procedure
requires the use of a fluorescence spectrophotometer.
The extraction of chromatin proteins starts with the isolation of a good nuclear
fraction. Nuclear pellets and chromatin should be extracted 1 day before the
laboratory period, if DNA, RNA, and both histone and nonhistone proteins are
to be separated.