Recombinant Polynucleosomes H3.3 (K27M)

Contents

Human Recombinant Polynucleosomes H3.3 (K27M) (20 µg protein + 24 µg DNA) are supplied in 10 mM Tris pH 8.0, 1 mM EDTA, 2 mM DTT, 20% glycerol. Please refer to product insert upon arrival for lot-specific concentration.

Background

In vivo, the nucleosome is the basic structural unit of chromatin. It is comprised of about 146 bp of DNA wrapped around a core of eight histones of four different types: H2A, H2B, H3 and H4. Histones are subject to posttranslational modifications, such as methylation, acetylation, phosphorylation and mono-ubiquitination.  Histone modifications influence multiple chromatin-templated processes such as gene transcription, DNA repair and recombination. Besides the “major“ histones, there are some histone variants in specific regions of chromatin or in specific cell types. Histone variants are involved in multiple processes including chromosome segregation, DNA repair, transcriptional regulation and mRNA processing. Histone H3.3 point mutations (K27 and G34) are found in 1/3 of pediatric glioblastomas. Up to 78% of diffuse intrinsic pontine gliomas (DIPGs) carry K27M and 36% of non-brainstem gliomas carry either K27M or G34R/V mutations. High- frequency mutation of histone H3 to K36M is observed in chondroblastomas, as is the mutation G34W/L in giant cell tumors of bone, diseases of adolescents and young adults. Histone H3.3 mutations drive pediatric glioblastoma through upregulation of MYCN. 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.

Application Notes

Recombinant Polynucleosomes H3.3 (K27M) are suitable as substrate for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling.

Protein Details

Recombinant Polynucleosomes H3.3 (K27M), 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) with a point mutation Lys27Met, and H4 that includes amino acids 1-103 (end) (accession number NM_003548). Plasmid pG5E4 contains 9 of 5S rDNA nucleosome positioning sequences of L. variegatus, 5 of GAL4 binding sites and E4 promoter. Every 5S rDNA can wrap one histone octamer to form a nucleosome. 5 of GAL4 binding sites and E4 promoter can wrap histone octamers to form dinucleosomes. A pG5E4 plasmid can warp histone octamers to form 11 nucleosomes. The recombinant protein is >95% pure by SDS-PAGE.

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