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Histone H3.2 Human, Recombinant |
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Description:
Histone H3 combines with Histone H4 to form the H3/H4 tetramer. Two H2A/H2B heterodimers interact with an H3/H4 tetramer to form the histone octamer (1,2). It is also modified by various enzymes and can act as a substrate for them. These modifications have been shown to be important in gene regulation.
Histone H3.2, an H.3 variant that is found in all eukaryotes except budding yeast, is replication dependent and is associated with gene silencing (3).
Protein Sequence:
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKS
TELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKR
VTIMPKDIQLARRIRGERA (Genbank accession number: Q71DI3)
Source:
An E.coli strain that carries a plasmid encoding the cloned human histone H3.2 gene, HIST2H3A or HIST2H3C. (Genbank accession number: BC130637)
Reaction & Storage Conditions
Concentration:
1 mg/ml
Storage Conditions:
20 mM sodium phosphate
300 mM NaCl
1 mM DTT
1 mM EDTA
pH 7.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- The protein concentration (1 mg/ml, 66 µM) is calculated using the molar extinction coefficient for Histone H3.2 (3960) and its absorbance at 280 nm (4,5). 1.0 A280 units = 3.9 mg/ml
Synonym: Histone H3/m, H3/o
Gene Synonym: H3F2, H3FM
FAQs
- Are the histones fusion proteins or tagged proteins?
- Do the histones need to be reconstituted?
- What are the recommended histone storage conditions?
- Can the histones be used as substrates for protein modification enzymes? Which ones?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
SDS-PAGE::
0.5, 1.0, 2.0, 5.0, 10.0 μg of Histone H3.2 Human, Recombinant were loaded on a 10-20% Tris-Glycine SDS-PAGE gel and stained with Coomassie Blue. The calculated molecular weight is 15256.82 Da. Its apparent molecular weight on 10-20% Tris-Glycine SDS-PAGE gel is ~17 kDa.
Mass Spectrometry::
The mass of purified Histone H3.2 Human, Recombinant is 15258.06 Da as determined by ESI-TOF MS (Electrospray Ionization-Time of Flight Mass Spectrometry). The average mass calculated from primary sequence is 15256.82 Da. This confirms the protein identity as well as the absence of any modifications of the histone.
N-terminal Protein Sequencing::
Protein identity was confirmed using Edman Degradation to sequence the intact protein.
Enzyme Modification: - G9a Methyltransferase: After incubation of a 25 µl reaction for 10 minutes at 37°C, 1 unit of G9a Methyltransferase (NEB #M0235) transfers 0.5 pmols of methyl group to Histone H3.2 Human, Recombinant.
- SET7 Methyltransferase: After incubation of a 25 µl reaction for 10 minutes at 37°C, 1 unit of SET7 Methyltransferase (NEB #M0233) transfers 1 pmols of methyl group to Histone H3.2 Human, Recombinant.
Protease Assay::
After incubation of 10 µg of Histone H3.2 Human, Recombinant with a standard mixture of proteins for 2 hours at 37°C, no proteolytic activity could be detected by SDS-PAGE.
Exonuclease Assay: Incubation of a 50 µl reaction containing 10 µg of Histone H3.2 Human, Recombinant with 1 µg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/µg) for 4 hours at 37°C released < 0.1% of the total radioactivity.
Endonuclease Assay: Incubation of a 50 µl reaction containing 10 µg of His
tone H3.2 Human, Recombinant with 1 µg of ϕX174 RF I (supercoiled) plasmid DNA for 4 hours at 37°C resulted in < 5.0% conversion to RF II form (nicked circle) as determined by agarose gel electrophoresis.
SDS-PAGE analysis of Histone H3.2 Human, Recombinant. Lane 1 & 7: NEB Protein Ladder (NEB #P7703), Lane 2 thru 6: 0.5-10.0 µg Histone H3.2 Human, Recombinant (Please see Quality Control section for more information)
ESI-TOF Analysis of Histone H3.2 Human, Recombinant.
References
- Kornberg, R.D. (1977) Annu. Rev. Biochem., 46, 931-954.
- van Holde, K.E. (1989) Chromatin, 1-497.
- Hake, S.B. et al. (2006) J. Biol. Chem., 281, 559-568.
- Gill, S.C. and von Hippel, P.H. (1989) Anal. Biochem., 182, 319-326.
- Pace, C.N. et al. (1995) Protein Science, 4, 2411-2423.
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