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.

Life Sensors的泛素和泛素链泛素是一种小的多肽，通过其C端与目标蛋白上的赖氨酸的ε-氨基缀合。这种缀合称为单泛素化。随后，可以利用遍在蛋白表面上的七个赖氨酸残基（K6，K11，K27，K29，K33，K48，K63）中的任何一个，将额外的遍在蛋白部分与初始遍在蛋白结合。这种泛素链形成称为多泛素化。泛素的酶结合是通过一系列酶进行的，即泛素激活酶E1，泛素结合酶E2和泛素连接酶E3。蛋白质的泛素化在细胞中是可逆的。单泛素化和多泛素化的链均被去泛素化酶催化的水解作用裂解 （DUB）。LifeSensors在遍在泛素途径的所有水平的酶（包括E1，E2和E3酶和DUB）上都有丰富的经验。LifeSensors对蛋白质纯化的广泛了解使我们能够生产大约30种E2酶，20种E3酶和35个DUB，并具有定制表达的能力。此外，我们广泛的检测方法简化了确认酶活性和确定E2-E3对兼容性的过程，并确保您可以选择最能解决希望探索的特定，关键生物学问题的酶。LifeSensors已为您的研究开发了多种泛素衍生物，包括用于结合相互作用研究的DUB耐药性聚泛素链，用于DUB分析和筛选的泛素结合物（荧光团和报告酶），选择性泛素链（二，三和四环素）泛素链）用于DUB特异性和结合测定，位点特异性赖氨酸泛素突变体用于E3连接酶和DUB选择性测定。