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Recombinant Proteins & Enzymes

Recombinant Mononucleosomes H3.3 (K36M) - biotin

Catalog No: 81007 Format: 20 µg $415 Buy Now
Expressed In: E. coli Protein Species: Human

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for Recombinant Mononucleosomes H3.3 (K36M) - biotinContents

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated (20 µg protein + 20 µg DNA) are supplied in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT and 20% glycerol. Please refer to product insert for lot-specific concentration.

for Recombinant Mononucleosomes H3.3 (K36M) - biotinBackground

In vivo, the nucleosome is the basic structural unit of chromatin. It consists 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, mono-ubiquitination, tec. 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 were involved in multiple biology 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 mutations in histone H3 include K36M in chondroblastomas and 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. 

for Recombinant Mononucleosomes H3.3 (K36M) - biotinApplication Notes

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated are suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling.

for Recombinant Mononucleosomes H3.3 (K36M) - biotinProtein Details

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated consist of a 167 bp of 601 DNA with 5’ biotin tag and two molecules each of histones H2A that includes amino acids 1-130 (end) (accession number NP_003503.1), H2B that includes amino acids 1-126 (end) (accession number NP_003509.1), H3.3 that includes amino acids 1-136 (end) (accession number NP_002098.1) with a point mutantion Lys36Met, and H4 that includes amino acids 1-103 (end) (accession number NP_003539.1). All of these histones were expressed in E. coli cells. The molecular weight of histone octamer is ~108 kDa. The recombinant protein is ≥95% by SDS-PAGE gel. 

 
Streptavidin pull-down for Recombinant Mononucleosomes H3.3 (K36M) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3.3 (K36M) - biotin.
Recombinant Mononucleosomes H3.3 (K36M) - biotinylated were pulled down by streptavidin beads. Input nucleosomes (Lane 2) and the nucleosomes (Lane 3) pulled down by streptavidin were run on a 13% SDS-PAGE gel and stained with Coomassie blue.  The SDS-PAGE gel result shows that almost all of biotinylated nucleosomes H3.3 (K36M) are pulled down by streptavidin beads. * indicates streptavidin. 

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated DNA gel.

Recombinant Mononucleosomes H3.3 (K36M) - biotin DNA gel.
Recombinant Mononucleosomes H3.3 (K36M) - biotin were run on a 2% agarose gel and stained with ethidium bromide. Lane 1: DNA marker. Lane 2: 601 DNA. Lane 3: Intact monnucleosomes H3.3 (K36M). Intact mononucleosomes H3.3 (K36M) migrated much higher than free 601 DNA.  The agarose gel result shows almost all of 601 DNA wrap histone octamers to form nucleosomes. 

Western Blot analysis for Recombinant Mononucleosomes H3.3 (K36M)

Western Blot analysis for Recombinant Mononucleosomes H3.3 (K36M)
 0.5 µg NSD2-SET (Cat. No. 31476) was incubated with 2 µg wild type Mononucleosomes H3.3 or K36M mutant Mononucleosomes H3.3, respectively, in 30 µl reaction system containing 50 mM Tris-HCl, pH 8.6, 0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP, 50 µM SAM for 3 hr at room temperature. 6 µl reaction products were loaded and run on a 12.5% SDS-PAGE gel. Western blot was used to detect the generation of reaction products (Anti-H3K36me2, Cat. No. 39891).  The Western blot result shows that K36M mutation inhibits the activity of NSD2-SET. 

for Recombinant Mononucleosomes H3.3 (K36M) - biotinStorage

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 Mononucleosomes H3.3 (K36M) - biotinGuarantee

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 Mononucleosomes H3.3 (K36M) - biotin
 

Data Thumbnails

Streptavidin pull-down for Recombinant Mononucleosomes H3.3 (K36M) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3.3 (K36M) - biotin.
(Click to enlarge and view details).

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated DNA gel.

Recombinant Mononucleosomes H3.3 (K36M) - biotinylated DNA gel.
(Click to enlarge and view details).

Western Blot analysis for Recombinant Mononucleosomes H3.3 (K36M)

Western Blot analysis for Recombinant Mononucleosomes H3.3 (K36M)
(Click to enlarge and view details).