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  Section: Biotechnology Methods » Molecular Biology
 
 
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Extraction and Electrophoresis of Histones

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Chromosomes


Morphotype Activated chromatin Euchromatin Nonactivated chromatin, Facultative and obligate heterochromatin, euchromatin
Structural organization Less condensed, unfolding of functional domains (2040 kbp) exhibit Highly condensed
DNA methylation (CG sites) DNase-I-sensitive sites mv-sites unmethylated mv-sites methylated
Nucleosomes DNase-I-sensitive sites DNase I resistant
Histones H1-deprivated; core histones highly acetylated H1-enriched; association with special H1 isofores, e.g., H5; H2A/H2B underacetylated; eventually H2A modified by ubiquitin
HMG 14/17 Present Absent
HMG 1/2 Present Absent
Transcription RNAP/RNP Presence of RNAP and RNP depends on the actual transcription state No
TABLE 1. Properties of chromatin

Materials
  • Saline citrate (1/10 SSC)
  • 1.0 N H2SO4
  • Refrigerated preparative centrifuge
  • Absolute ethanol
  • β-mercaptoethanol
  • 0.01 M sodium phosphate buffer, pH 7.0 + 1% (w/v) SDS + 0.1% (v/v)
  • β-mercaptoethanol
  • 10% acrylamide gels (10%T:5%C) with 0.1% (w/v) SDS
  • 7% (w/v) acetic acid
  • 0.25% Coomassie Blue
Procedure
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.
  1. Stir the solution on ice and slowly add ¼ volume of cold 1.0 N H2SO4. Continue stirring for 30 minutes.
  2. Centrifuge the suspension at 12000 xg for 20 minutes at 4°C. Save the supernatant. For maximum yield, break up the pellet, resuspend in fresh, cold 0.4 N H2SO4, re-extract, centrifuge, and add the resulting supernatant to the first.
  3. Add 4 volumes of cold absolute ethanol to the supernatant and store for 24 hours at –10° C to precipitate the histone-sulfates.
  4. Collect the precipitate by centrifugation at 2000 xg for 30 minutes.
  5. Decant as much of the alcohol as possible, and resuspend the pellet in cold absolute ethanol.
  6. Centrifuge at 10000 xg for 15 minutes.
  7. 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% β-mercaptoethanol; the final volume should contain approximately 300 mg of protein in 100 mL of buffer.
  8. Prepare the electrophoresis chamber with a 10% acrylamide gel with 0.1% SDS.
  9. Add separately 25 µL of the dissolved protein and 25 µL of protein standards to:
    50 µL of 0.1% SDS, 0.1% β-mercaptoethanol in buffer
    5 µL of β-mercaptoethanol
    1 µL of 0.1% bromophenol Blue in water
  10. Mix thoroughly and apply the histone extract and protein standards to separate wells of the electrophoresis gel.
  11. Separate the proteins in the anode direction (anionic system).
    • The addition of SDS anions to the proteins results in negatively charged proteins, which will separate according to molecular weight.
    • Electrophoresis is carried out in the standard manner. The buffer utilized is Laemmli.
    • 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).
  12. Stain the gels with 0.25% Coomassie Blue for 2 hours.
  13. Destain and store in 7% acetic acid.
  14. Scan the gels and determine the molecular weights of each component.
Notes
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 nonhistones immediately, while the dissociation of chromatin in salt solutions will require further purification. In either event, the histones themselves are subdivided into 5 major types, designated as H1, H2, H3, H4, and H5. H2 dissociates into 2 peptides, which are thus designated as H2A and H2B. The classification of histones is based on their electrophoretic mobility.

Nonhistone proteins can also be extracted and separated by electrophoresis. Whereas histones have only 5 major types, nonhistones 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. 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 polytene chromosomes.
 
     
 
 
     




     
 
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