Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College
LEVEL II
Materials
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
TCA (Trichloroacetic acid)
Tissue homogenizer
Cheesecloth
Refrigerated preparative centrifuge
Bradford or Lowry protein assay
UV spectrophotometer (Optional)
Procedure 3
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 3,500 xg for 20 minutes.
Resuspend the nuclear pellet in 80 ml of cold 0.25 Sucrose containing 0.0033 M Calcium Acetate. 4
Obtain three 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 40,000 xg for 60 minutes.
Using the resulting nuclear pellet is just above the dense sucrose layer. It is used to extract chromatin proteins. Carefully remove the supernatant above the pellet with a pipet. Then, insert the pipet 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 7,700 xg for 10 minutes.
Repeat step 7 to thoroughly wash the nuclei and then wash 2x 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 procedure.
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 OD
. A 10 ug/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 spectrophotometer.
Measure by non-destructive fluorometry. Histones can be detected by an excitation wavelength of 280 nm and a fluorescence measurement at 308 nm. Non-histones 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.
Notes
The extraction of chromatin proteins starts with the isolation of a good nuclear fraction. Nuclear pellets and chromatin should be extracted one day before the laboratory period. If DNA, RNA, and both Histone and Non-Histone proteins are to be separated, begin the procedure approximately three working days prior to lab.
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