Recombinant Mononucleosomes (H1.2)

Catalog No: 81272 Format: 20 µg $440 Buy
Catalog No: 81972 Format: 1 mg $5,500 Buy

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Expressed In: E. coli Protein Species: Human

Contents

A representative Technical Data Sheet (TDS) is provided here. Please refer to the lot-specific TDS you will receive with your order for the lot-specific buffer contents and protein concentration.

Background

Histones are basic nuclear proteins responsible for nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. In vivo, histones are wrapped around by DNA in chromatin. Therefore, nucleosomes are more physiologically relevant substrates than histones and histonederived peptides for in vitro studies. More importantly, some histone methyltransferases are significantly more active, as well as specific, when using nucleosomal substrates in HMT assays, such as DOT1L and NSD family enzymes. Nucleosomes are also widely used in histone methyltransferase screening assays to identify small molecular inhibitors for drug discovery. The called linker histone, H1, binds to linker DNA between nucleosomes forming the macromolecular structure known as the chromatin fiber. So it is necessary for the condensation of nucleosome chains into higher-order structured fibers. It can also act as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing and DNA methylation. Histone H1.2 is a replication-dependent histone that is a member of the histone H1 family.

Application Notes

Recombinant Mononucleosomes (H1.2) are suitable for use as substrate of enzymatic assay or other biochemical assay.

Protein Details

Recombinant Mononucleosomes (H1.2) consist of a 204 bp of 601 DNA (48 bp and 10 bp on 5’ and 3’ of 146 bp core DNA, respectively) and two molecules each of histones H2A that includes amino acids 1-130 (end) (accession number NM_003512), H2B that includes amino acids 1-126 (end) (accession number NM_003518), H3.1 that includes amino acids 1-136 (end) (accession number NM_003529), and H4 that includes amino acids 1-103 (end) (accession number NM_003548), and histone H1.2 that includes amino acids 1-213 (end) (accession number NP_005310.1). All of these histones were expressed in E. coli cells. The molecular weight of histone octamer and histone H1.2 are 108 kDa and 21.4 kDa, respectively.

 

Recombinant Mononucleosomes (H1.2) DNA gel
Recombinant Mononucleosomes (H1.2) were run on a 2% agarose gel and stained with ethidium bromide. Lane 1: DNA marker. Lane 2: 601 DNA. Lane 3: Intact monnucleosomes (H1.2). Intact mononucleosomes (H1.2) migrated much higher than free 601 DNA. The agarose gel result shows almost all of 601 DNA wrap histone octamers to form nucleosomes.

Recombinant Mononucleosomes (H1.2) protein gel
13% SDS-PAGE with Coomassie Blue staining
Purity: ≥95%

Western blot assay for Recombinant Mononucleosomes (H1.2)
2 µg Recombinant Mononucleosomes were incubated with 0, 12.5, 25, 50 ng DOT1L (Cat. No. 31474) respectively in reaction buffer containing 50 mM Tris-HCl pH 8.6, 0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP and 50 µM SAM for 2 hr at room temperature. Half of the reactions were run on a 12.5% SDS-PAGE gel and detected with H3K79me1 antibody (Cat. No. 39921) and H4 antibody (Cat.No. 39269), respectively. DOT1L protein and Recombinant Histone Octamers were used as controls. H4 was detected as the loading control. The result shows that mononucleosomes are better than octamers as substrates for DOT1L.

Storage

Recombinant proteins in solution are temperature sensitive and must be stored at -80°C to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

Guarantee

This product is guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic