Specifications

Phosphatase and TENsin homolog (PTEN) negatively regulates the phosphatidylinositol-3-kinase/AKT pathway and is one of the most frequently mutated genes in human cancer. Monoubiquitylation of PTEN at lysine 13 and lysine 289 results in increased nuclear localization of PTEN where it is implicated in phosphatase-independent functions including regulation of the activity and stability of p53. This monoubiquitylated peptide can be used for deubiquitination 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 monoubiquitinated peptide as a DUB substrate measures cleavage of an isopeptide bond and allows determination of the influence of sequence proximal to the ubiquitinated lysine on specificity. Profiling activity of a panel of DUBs against a monoubiquitinated histone H2B peptide and a monoubiquitinated PTEN peptide reveals interesting differences in specificity:

Assessment of DUB activity using monoubiquitylated peptides as a substrates. 2µM monoubiquitylated histone H2B peptide (red), 2µM monoubiquitylated 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 monoubiquitylated 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.

Custom monoubiquitylated peptides can be generated from the sequence of your choosing for use as DUB substrates or in ubiquitin binding studies. Various affinity tags, fluorescent labels and mutant ubiquitins are available to facilitate different assay modalities. Please contact us for a quote or more information.

Info

Peptide	Ac-PTEN[5-21](Ub)-CONH2
Sequence	Ac-IKEIVSRNK(ubiquitin)RRYQEDGF-NH2
Purity	>95% by HPLC
Molecular Weight	10,726 Da by MS
Physical State	Lyophilized powder
Quantity	50 µg
Solubility	at least 1mg/ml in aqueous buffer
Storage	Dry, -20 °C. Solution, -80 °C. Avoid repeated freeze/thaw cycles

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。