• Description

• Ordering information

• Features

• Applications

• References

• Protocols

 

Description.

Yorkshire Bioscience offers high-quality histones isolated from calf thymus for use as substrates in protein kinase and methylase reactions.

Histones are basic proteins that are found complexed with DNA in cell nuclei of eukaryotic cells. Bacteria do not have histones. However, the DNA of the genus Thermoplasma (in the domain Archaea) is surrounded by a highly basic DNA-binding protein which strongly resembles the eukaryotic histones.

A chromosome contains five types of histones: H1 (or H5), H2A, H2B, H3 and H4.  H1 and its homologous protein H5 are involved in higher-order structures.  The other four types of histones associate with DNA to form nucleosomes.  H1 (or H5) has about 220 residues.  The other types of histones are smaller, each consisting of 100-150 residues.

An important feature about histones is that they contain a few lysine (K) residues at the N terminus.  Under normal cellular conditions, the R group of lysine is positively charged, which allows it to interact with the negatively charged phosphates in DNA.  The positive R group of lysine may be neutralized by acetylation, reducing the binding force between histones and DNA.  This mechanism has been demonstrated to play a major role in the regulation of gene transcription.

 Article for review: Origin of H1 linker histones - FASEB J., 2001.

  

Ordering information:

Cat.#	Product	Pack size	Price
AVH-T50	Histone Total	50 mg	£300
AVH-HI5	Histone H1	5 mg	£200
AVH-HIIB5	Histone H2A	5 mg	£200
AVH-HIIB5	Histone H2B	5 mg	£200
AVH-HIII5	Histone H3	5 mg	£200
AVH-HIV5	Histone H4	5 mg	£200

 All histones are also available in bulk quantities up to 100 g of each.

Shipment: ambient temperature.

Store in dry dark place at -20 ºC for more than 24 months.

 

Features:

Name of Histone	Total	H1	H2A	H2B	H3	H4
Molecular weight, Da	Mixture of individual histones	21500	14004	13774	15324	11282
Molar absorption coefficient (280 nm)	n/a	1280	3840	6400	3960	5120
A280, 1 mg/ml	0.3	0.06	0.275	0.465	0.258	0.456
A230, 1 mg/ml	>2.0	>2.0	>2.0	>2.0	>2.0	>2.0
Purity	n/a	> 95% as determined by SDS-PAGE
Appearance	White lyophilized powder. Lyophilized from water
Protein content	>0.9 mg of protein / mg of lyophilized powder
Stability	At least 5 years at room temperature
Appearance when dissolved in water	Clear, colourless

 

 

Applications:

• Substrate for protein kinase^{1,2, 4}, deacetylase^5,6 and methylase³
• Experiments on DNA-protein interactions

 

References:

1. J. Biol. Chem., 1998,  273 (2): 25893–25902.

2. J. Biol. Chem., 2000, 275 (4): 2431-2438.

3. J. Biol. Chem., 2001, 276 (27): 25309-25317.

4. J. Biol. Chem., 1996, 271(18)10461-10469

5. Nature, 2000,  403: 41.

6. Curr. Opin. Cell Biol., 2000, 12: 326.

 

 

Protocols:

In Vitro Assay of Protein Kinase Activity -- Samples of the recombinant kinases were incubated in phosphorylation buffer (25 mM Hepes, 5 mM MgCl₂, 5 mM MnCl₂, 0.5 mM dithiothreitol) containing 10 µM [-³²P]ATP (100 µCi/ml) for 10 min or 30 min for different preparations at room temperature. Substrates (histones) were added to a final concentration of 0.1 - 0.25 mg/ml. Reaction products were separated by SDS-PAGE (16% gels) and visualized by autoradiography of the dried gels.

In Vitro Methyltransferase Assays -- 50 µl of reaction mixture containing substrates (a mixture of histones H1, 2A, 2B, 3, and 4; and single H1, H2A, and H3), enzymes, and 250 nCi of S-adenosyl-[methyl-¹⁴C]-L-methionine in methylase activity buffer (50 mM Tris, pH 8.5, 20 mM KCl, 10 mM MgCl₂, 10 mM -mercaptoethanol, 250 mM sucrose) was incubated for 60 min at 37 °C. The reaction products were separated by 15% SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue R-250 staining and by autoradiography of the dried gels.