Assays & Kits

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1),  a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2~Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2~Ub to the substrate, leading to its mono- or poly-ubiquitination. The SMAD ubiquitination regulatory factor 1 (SMURF1) is a HECT-type E3 Ub ligase that regulates TGF-β/BMP pathways via ubiquitination of key signal transducers (SMAD1, SMAD2, or SMAD5), or TGF-β receptor I. SMURFs play a critical role in cell-type specification, tissue and organ development by regulating planar cell polarity signaling and convergent extension. SMURFs can also accele

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1),  a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2~Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2~Ub to the substrate, leading to its mono- or poly-ubiquitination. The SMAD ubiquitination regulatory factor 2 (SMURF2) is a HECT-type E3 Ub ligase that regulates TGF-β/BMP pathways via ubiquitination of key signal transducers (SMAD1, SMAD2, or SMAD5), or TGF-β receptor I. SMURFs play a critical role in cell-type specification, tissue and organ development by regulating planar cell polarity signaling and convergent extension. SMURFs can also accele

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1),  a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2~Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2~Ub to the substrate, leading to its mono- or poly-ubiquitination. The von Hippel-Lindau protein (VHL) interacts with Elongins B and C, Cullin 2, and Ring Box Protein 1 (Rbx1) to form the functional E3 Ub ligase complex where it functions as a substrate recognition entity. Most of the tumor-derived mutations within VHL disrupt its tumor suppressor role by compromising its substrate receptor function and generally whole VHL complex Ub ligase activit

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1),  a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2~Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2~Ub to the substrate, leading to its mono- or poly-ubiquitination. The X-linked inhibitor of apoptosis (XIAP) protein is a RING-containing E3 Ub ligase which has the ability to directly regulate caspases and suppress apoptotic cell death pathways. An increased expression level of XIAP has been shown for many cancer types and is associated with cancer cell migration. Like most E3 ligases, XIAP ubiquitinates itself. The XIAP intrachain TR-FRET Assay

The PARPtrap™ Combo Assay Kit for PARP1 and PARP2 is a kit designed to measure DNA complex formations of PARP1 and PARP2 enzymes in a high throughput screening assay using fluorescence polarization (FP). The key to the PARPtrap™ Combo Assay Kit are the fluorescent-labeled DNA probes recognized by PARP1 and PARP2. In the absence of ribosylation, PARP1/2 binds to the DNA fluorescent probe, forming a large complex and resulting in the emission of highly polarized light. However, after autoribosylation, PARP1/2 dissociates from the oligonucleotide duplex, which then rotates freely, emitting less polarized light (Fig. 1). Addition of a PARP inhibitor results in trapping of PARP1/2 to the fluorescent-labeled DNA, and increases the FP signal in a dose dependent manner. The combo kit is particularly useful to directly and specifically compare, in a single assay, the effect and potency of a compound on PARP1 and PARP2. The PARPtrap™ Combo Assay Kit for PARP1 and PARP2 is a homogeneous fluore

The CBX1 Inhibitor Screening Assay Kit is designed to measure the inhibition of CBX1 binding to its substrate. The kit comes in a convenient AlphaLISA® format, with enough biotinylated peptide substrate, assay buffer, detection buffer and purified GST-tagged CBX1 chromodomain to perform a total of 384 enzyme reactions. The key to the kit is the specific binding of the CBX1 chromodomain to the methylated-peptide substrate. With this kit, only three simple steps on a microtiter plate are required. First, a sample containing CBX1 and an inhibitor of choice is incubated with the biotinylated substrate for one hour. Next, acceptor beads are added, then donor beads, followed by reading the Alpha-counts.

The GITR:GITRL TR-FRET Assay is designed to measure the inhibition of GITR binding to GITRL in a homogeneous 384 reaction format. This FRET-based assay requires no time-consuming washing steps, making it especially suitable for high throughput screening applications. The assay procedure is straightforward and simple; a sample containing biotinylated GITR, GITRL, anti-His Tb donor, dye-labeled acceptor, and an inhibitor is incubated for two hours. Then, the fluorescence intensity is measured using a fluorescence reader.

The USP7 Inhibitor Screening Assay Kit is designed to measure USP7 activity for screening and profiling applications. The USP7 Inhibitor Screening Assay Kit comes in a convenient format, with a 96-well plate, fluorogenic, ubiquitinated substrate, assay buffer, and purified USP7 enzyme for 96 enzyme reactions. USP7 is incubated with a sample containing assay buffer and Ub-AMC Substrate, and then the plate is read, measuring fluorescence using a fluorescence reader.

The CD40:CD40L[Biotinylated] Inhibitor Screening Assay Kit is designed for screening and profiling inhibitors of CD40:CD40L signaling. This kit comes in a convenient 96-well format, with biotin-labeled CD40, purified CD40L, streptavidin-labeled HRP, and assay buffer for 100 binding reactions. The key to this kit is the high sensitivity of detection of biotin-labeled CD40 by streptavidin-HRP. Only a few simple steps on a microtiter plate are required for the assay. First, CD40L is coated on a 96-well plate. Next, CD40 is incubated with CD40L on the plate. Finally, the plate is treated with streptavidin-HRP followed by addition of an HRP substrate to produce chemiluminescence, which can be measured using a chemiluminescence reader.