Specifications

Monoubiquitylation of lysine 120 on human histone H2B is associated with transcriptional activity. The RNF20/40 complex functions as the E3 ligase and UbcH6 as the E2 conjugating enzyme to monoubiquitinate this site to stimulate HOX gene expression. Monoubiquitylation of lysine 120 increases histone chaperone FACT function resulting in transcript elongation and the generation of longer transcripts. This monoubiquitylated peptide can be used for deubiquitylation assays or to study ubiquitin binding domain recognition.

In contrast to traditional DUB substrates, like ubiquitin-AMC and ubiquitin-rhodamine, which measure cleavage of amide bonds, utilization of a monoubiquitylated peptide as a DUB substrate measures cleavage of an isopeptide bond and allows determination of the influence of sequence proximal to the ubiquitylated lysine on specificity. Profiling activity of a panel of DUBs against a monoubiquitinated histone H2B peptide and a monoubiquitylated PTEN peptide reveals interesting differences in specificity:

Peptide Ac-H2B[110-125](Ub)-CONH2 Sequence Ac-AVSEGTKAVTK(ubiquitin)YTSSK-NH2 Purity >95% by HPLC Molecular Weight 10,245 Da by MS Physical State Lyophilized powder Quantity 50 µg Solubility At least 1 mg/mL in aqueous buffer Storage Dry, -20 °C. Solution, -80 °C.Avoid repeated freeze/thaw cycles

Assessment of DUB activity using monoubiquitinated peptides as a substrates. 2µM monoubiquitinated histone H2B peptide (red), 2µM monoubiquitinated PTEN peptide (blue), and 2µM ubiquitin-rhodamine were incubated with 50nM of each indicated DUB for 30 minutes at 37ºC. Isopeptidase cleavage of ubiquitin from the monoubiquitinated peptides was assessed by LC-MS and is represented as the percent substrate cleaved. Cleavage of ubiquitin-rhodamine was measured as the increase in rhodamine fluorescence at 535nm and is represented as the rate (RFU/sec) relative to USP2c.

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