DNA & Clones

The Ub-CHOP2-Reporter Deubiquitylation Assay consists of ubiquitin fused to a reporter enzyme, as well as a separate reagent substrate for the reporter enzyme. When fused to ubiquitin, the reporter is rendered catalytically inactive. Following cleavage of the Ub-reporter system by the isopeptidase, the free (and now active) reporter subsequently acts upon its substrate. Thus, in this coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity.-\n<h3><strong>Contents</strong></h3>-\n-- Ub-CHOP2-Reporter System: 1 x 375 &#0956;L (4 &#0956;M)-\n-- USP2 (Control Isopeptidase): 2 x 10 &#0956;L (2 &#0956;M)-\n-- Reporter Substrate 2: 1 x 30 &#0956;L (25 &#0956;M)-\n-- Control Fluorophore 2: 1 x 20 &#0956;L (1 mM)

The NEDD8-CHOP-Reporter DeNEDDylation Assay Kit consists of NEDD8 fused to a reporter enzyme and the reporter enzyme substrate. When fused to NEDD8, the reporter is rendered catalytically inactive. Following cleavage of the NEDD8-reporter system by isopeptidase activity, the free (and now active) reporter enzyme subsequently acts upon its substrate. Thus, in this coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity.-\n<h3><strong>Contents</strong></h3>-\n-- NEDD8-CHOP2-Reporter (Reporter System): 1 x 375 &#0956;L (4 &#0956;M)-\n-- Den1 (Control Isopeptidase): 2 x 10 &#0956;L (2 &#0956;M)-\n-- Reporter Substrate 2: 1 x 30 &#0956;L (25 &#0956;M)-\n-- Control Fluorophore 2: 1 x 20 &#0956;L (1 mM)

The ISG15-CHOP2-Reporter DeISGylation Assay consists of ISG15 fused to a reporter enzyme, as well as a separate reagent substrate for the reporter enzyme. When fused to ISG15, the reporter is rendered catalytically inactive. Following cleavage of the ISG15-reporter system by the isopeptidase, the free (and now active) reporter subsequently acts upon its substrate. Thus, in the coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity.-\n<h3><strong>Contents</strong></h3>-\n-- ISG15-CHOP2-Reporter System: 1 x 375 &#0956;L (4 &#0956;M)-\n-- PLpro (Control Isopeptidase): 2 x 10 &#0956;L (2 &#0956;M)-\n-- Reporter Substrate 2: 1 x 30 &#0956;L (25 &#0956;M)-\n-- Control Fluorophore 2: 1 x 20 &#0956;L (1 mM)

The pY-SUMOstar vector allows for the intracellular expression of SUMOstar fusion proteins in <em>Saccharomyces cerevisiae</em>. SUMOstar fusion constructs are not processed <em>in vivo</em>, allowing for the same enhancement of protein expression, solubility, and stability afforded by SUMOpro.Protein expression is driven by the <em>S. cerevisiae</em> copper metallothionein (CUP1) promoter, which is both tightly regulated and highly inducible.  The plasmid is maintained by the 2-micron element and selected for by the restoration of L-tryptophan biosynthesis in Trp auxotrophs.

The pY-secSUMOstar vector enables expression and secretion of SUMOstar fusions in Pichia pastoris. Protein expression is driven by the primary alcohol oxidase (AOX1) promoter, which is both tightly regulated and highly inducible. The alpha mating factor (S. cerevisiae) targets the fusion protein to the secretory pathway, while both a FLAG and a His6 tag preceed SUMOstar for convenient purification. Chromosomal integration is accomplished by DNA linearization within the promoter using a variety of convient endonuclease restriction sites, while mult-copy integrants can be obtained through strain selection against increasing concentrations of the antibiotic Zeocin.

Salmon Sperm DNA was shared by sonication.DNA is dissolved in deionized distilled water, with a fragment size range of 300-700 base pairs.