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リコンビナントタンパク質・酵素

Recombinant Polynucleosomes (H3.3)

商品コード: 31468 Format: 20 µg ¥73,000 Buy Now
Expressed In: E. coli Protein Species: Human

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for Recombinant Polynucleosomes (H3.3)Contents

Recombinant Nucleosomes (H3.3) (20 µg protein + 24 µg DNA) is supplied in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT and 20% glycerol. Please refer to product insert upon arrival for lot-specific concentration.

for Recombinant Polynucleosomes (H3.3)Background

In vivo, histones are wrapped around by DNA in chromatin. Therefore, nucleosomes are more physiologically relevant substrates than histones and histone-derived 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.

Histone H3.1 and Histone H3.3 are the two main Histone H3 variants found in plants and animals. They are known to be important for gene regulation. Histone H3.1 and H3.3 have been shown to demonstrate unique genomic localization patterns thought to be associated with their specific functions in regulation of gene activity. Specifically, Histone H3.1 localization is found to coincide with genomic regions containing chromatin repressive marks (H3K9me3, H3K27me3 and DNA methylation), whereas Histone H3.3 primarily colocalizes with marks associated with gene activation (H3K4me3, H2BK120ub1, and RNA pol II occupancy). Deposition of the Histone H3.1 variant into the nucleosome correlates with the canonical DNA synthesis-dependent deposition pathway, whereas Histone H3.3 primarily serves as the replacement Histone H3 variant outside of S-phase, such as during gene transcription. Aberrant localization of these variants is also known to correlate with certain cancers.

for Recombinant Polynucleosomes (H3.3)Application Notes

Recombinant Polynucleosomes (H3.3) are suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling.

for Recombinant Polynucleosomes (H3.3)Protein Details

Recombinant Polyucleosomes (H3.3), Human, consist of 5000 bp of DNA (plasmid pG5E4) 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), H3.3 that includes amino acids 1-136 (end) (accession number NM_005324), and H4 that includes amino acids 1-103 (end) (accession number NM_003548). Plasmid pG5E4 includes 11 5S rDNA nucleosome positioning sequences of L. variegatus. All of these histones were expressed in E. coli . The molecular weight of the histone octamer is ~108 kDa. Every 5S rDNA can wrap one histone octamer to form a nucleosome. Recombinant polynucleosomes include at least 11 nucleosomes. The recombinant protein is >95% pure by SDS-PAGE.

 
Recombinant Polynucleosomes (H3.3) protein gel.

Recombinant Polynucleosomes (H3.3) protein gel.
Recombinant Polynucleosomes (H3.3) were run on a 12% SDS-PAGE gel and stained with Coomassie Blue.

Recombinant Polynucleosomes (H3.3) - DNA Gel-shift assay.

Recombinant Polynucleosomes (H3.3) protein DNA Gel-shift assay
Polynucleosomes (H3.3) and free plasmid DNA were run on 1% agarose gel and stained with ethidium bromide. Intact polynucleosomes migrate much higher than free DNA, thus the DNA resolves at a higher molecular weight when nucleosome-bound.

Recombinant Polynucleosomes (H3.3) HMT activity assay

Histone Methyltransferase (HMT) Assay comparing Recombinant Polynucleosomes (H3.3) and histone octamers as substrates.
2 µg Recombinant Polynucleosomes (H3.3) were incubated with 0 µg , 0.25 µg, 0.5 µg and 1 µg NSD2-SET (Cat# 31476) in reaction buffer for 3 hours at room temperature, respectively. Western Blot was used to detect the generation of reaction products (H3K36me2, Cat# 39891). NSD2-SET only and polynucleosomes only were used as negative control. The Western Blot result shows that polynucleosomes are more suitable substrate for NSD2 than histone octamers.

for Recombinant Polynucleosomes (H3.3)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.

for Recombinant Polynucleosomes (H3.3)Guarantee

This product is for research use only and is not for use in diagnostic procedures. This product is guaranteed for 6 months from date of arrival.

 

Technical Data Sheet

Recombinant Polynucleosomes (H3.3)
 

Data Thumbnails

Recombinant Polynucleosomes (H3.3) protein gel.

Recombinant Polynucleosomes (H3.3) protein gel, stained with Coomassie Blue.
(Click image to enlarge and see details).

Recombinant Polynucleosomes (H3.3) - DNA Gel-shift assay.

Recombinant Polynucleosomes (H3.3) - DNA Gel-shift assay.
(Click image to enlarge and see details).

Recombinant Polynucleosomes (H3.3) HMT activity assay

Recombinant Polynucleosomes (H3.3) HMT activity assay.
(Click image to enlarge and see details).