Exercise 10.5 - Extraction and Electrophoresis of Histones
Table 10.1 Properties of chromatin
Figure 10.3 Chromatin electrophoresis pattern
- Saline Citrate (1/10 SSC)
- 1.0 N HSO
- Refrigerated preparative centrifuge
- Absolute ethanol
- 0.01 M Sodium phosphate buffer, pH 7.0 + 1% (w/v) SDS + 0.1% (v/v) -mercaptoethanol
- 10% acrylamide gels (10%T:5%C) with 0.1% (w/v) SDS
- 7% (w/v) Acetic acid
- 0.25% Coomasie blue
- Dissolve crude chromatin in cold dilute saline citrate
(0.015 M NaCl + 0.001 M Sodium Citrate) to a final DNA
concentration of 500 µg/ml.
- Stir the solution on ice and slowly add 1/4 volume of cold
1.0 N HSO. Continue stirring for 30 minutes.
- Centrifuge the suspension at 12,000 xg for 20 minutes at 4°
C. Save the supernatant. For maximum yield, break
up the pellet, resuspend in fresh, cold 0.4 N HSO, re-extract, centrifuge, and add the resulting
supernatant to the first.
- Add 4 volumes of cold absolute ethanol to the supernatant
and store for 24 hours at -10° C to precipitate the
- Collect the precipitate by centrifugation at 2,000 xg for
- Decant as much of the alcohol as possible, and resuspend the
pellet in cold absolute ethanol.
- Centrifuge at 10,000 xg for 15 minutes.
- Collect the pellet and freeze dry for later analysis.
To continue with the electrophoresis, carefully weigh the histone
protein sample and dissolve in 0.01 M sodium phosphate buffer,
with a pH 7.0 and containing 1% sodium dodecyl sulfate and 0.1%
-mercaptoethanol; final volume should contain approximately
300 µg of protein in 100 µl of buffer.
- Prepare the electrophoresis chamber with a 10% acrylamide gel
with 0.1% SDS.
- Add separately 25 µl of the dissolved protein and 25 µl
of protein standards to:
50 µl of 0.1% SDS, 0.1% -mercaptoethanol in Buffer
5 µl of -mercaptoethanol
1 µl of 0.1% bromophenol blue in water
- Mix thoroughly and apply the histone extract and protein
standards to separate wells of the electrophoresis gel.
- Separate the proteins in the anode direction (Anionic
- The addition of SDS anions to the proteins results in
negatively charged proteins which will separate according to
- Electrophoresis is carried out in the standard manner
following the basic steps given in Chapter Four. The buffer
utilized is that of Laemmli, 7 0.025 M Tris-0.192
Glycine and 0.1% SDS, pH 8.3.
- Proteins are separated by a current of 3-4 mA per gel until the
bromophenol marker reaches the bottom of the tube (about 7 hours at
3 mA, and 4 hours at 4 mA).
- Stain the gels with 0.25% Coomasie Blue for 2 hours.
- Destain and store in 7% acetic acid.
- Scan the gels and determine the molecular weights of each
Preparation of a total histone fraction from nuclei is
normally accomplished by extraction with a dilute acid or a high
molarity salt solution. The acidic extraction removes histones
from DNA and non-histones immediately, while the dissociation of
chromatin in salt solutions will require further purification. In
either event, the histones themselves are subdivided into five
major types, designated as H1, H2, H3, H4 and H5. H2 dissociates
into two peptides, which are thus designated as H2A and H2B. The
classification of histones is based on their electrophoretic
Non-histone proteins can also be extracted and separated by
electrophoresis. Whereas histones have only 5 major types, non-
histones are extremely heterogeneous and up to 500 different
proteins have been identified from one cell type, while the major
proteins comprise less than 20 types.
The extraction of chromatin DNA was possible with the 7 M
urea - 3 M NaCl extraction performed in Exercise 10.4. Further
analysis of DNA will be undertaken as part of a later lab
exercise (on Transcription), and the DNA sample from this lab may
be kept lyophilized and frozen until that time.
For our current needs it is sufficient to note that the
genes are composed of DNA, and that various specific regions of
the DNA/Genetic information can be physically isolated to a
specific locus on a chromosome. This in turn is readily observed
and correlated with banding patterns, such as those in the fruit
fly poltytene chromosomes.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org