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Recombinant Proteins & Enzymes

Recombinant Mononucleosomes H3K14ac (EPL) - biotin

Catalog No: 81001 Format: 20 µg $445 Buy Now
Expressed In: E. coli Protein Species: Human

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Expressed Protein Ligation

Recombinant Mononucleosomes H3K14ac (EPL) - biotin has been generated using the patented Expressed Protein Ligation (EPL) technology. EPL can be used to generate methylated, acetylated or phosphorylated histones. In EPL, the histone globular domain is ligated to a peptide that contains the N-terminal histone tail with the desired site-specific modification. The ligation reaction maintains the native histone bonds, and the resulting recombinant protein more closely mimics natural histones.

for Recombinant Mononucleosomes H3K14ac (EPL) - biotinContents

Recombinant Mononucleosomes H3K14ac (EPL) - 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 upon arrival for lot-specific concentration.

for Recombinant Mononucleosomes H3K14ac (EPL) - biotinBackground

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. 

for Recombinant Mononucleosomes H3K14ac (EPL) - biotinApplication Notes

Recombinant Mononucleosomes H3K14ac (EPL) - biotinylated are suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling.

for Recombinant Mononucleosomes H3K14ac (EPL) - biotinProtein Details

Recombinant Mononucleosomes H3K14ac (EPL) - 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), H3.2 that includes amino acids 1-136 (end) (accession number NP_066403.2) with acetylation at lysine 14, 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. 

H3K14ac (Histone H3 acetyl Lys14) protein is generated using expressed protein ligation (EPL) technology. Truncated human Histone H3.2 is produced in E. coli and purified using FPLC. The purified protein is subsequently ligated to a N-terminal histone tail peptide containing acetyl lysine 14 via a native peptide bond. 

 
Streptavidin pull-down for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.
Recombinant Mononucleosomes H3K14ac (EPL) 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 Commassie blue. The SDS-PAGE gel result shows that almost all of biotinylated mononucleosomes H3K14ac are pulled down by streptavidin beads. * indicates streptavidin.

Recombinant Mononucleosomes H3K14ac (EPL) - biotin, DNA gel.

Recombinant Mononucleosomes H3K14ac (EPL) - biotin, DNA gel.
Recombinant Mononucleosomes H3K14ac (EPL) - biotinylated were run on a 2% agarose gel and stained with ethidium bromide. Lane 1: DNA marker. Lane 2: 601 DNA. Lane 3: Intact monnucleosomes H3K14ac. Intact mononucleosomes H3K14ac 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 H3K14ac (EPL) - biotin.

Western Blot analysis for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.
Unmodified nucleosomes (Lane 1) and Recombinant Mononucleosomes H3K14ac (EPL) - biotinylated (Lane 2) were detected with anti-H3K14ac antibody and anti-H4 antibody, respectively. H4 was detected as loading control. Only Recombinant Mononucleosomes H3K14ac (EPL) - biotinylated can be detected by anti-H3K14ac antibody.

for Recombinant Mononucleosomes H3K14ac (EPL) - 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 H3K14ac (EPL) - 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 H3K14ac (EPL) - biotin
 

Data Thumbnails

Streptavidin pull-down for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.

Streptavidin pull-down for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.
(Click to enlarge and view details).

Recombinant Mononucleosomes H3K14ac (EPL) - biotin, DNA gel.

Recombinant Mononucleosomes H3K14ac (EPL) - biotin, DNA gel.
(Click to enlarge and view details).

Western Blot analysis for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.

Western Blot analysis for Recombinant Mononucleosomes H3K14ac (EPL) - biotin.
(Click to enlarge and view details).