Functional Assays

The 3CL Protease (P252L) (SARS-CoV-2) Assay Kit is a 96-well homogeneous fluorogenic based assay designed to measure the activity of P252L mutated 3CL Protease for screening and profiling applications, with no time-consuming washing steps. 3CL inhibitor GC376 is also included as a control. 3CL inhibitor GC376 is also included as a control.  <img src="{{media url="wysiwyg/coronavirus/78834_pic.png"}}" alt="" width="551" height="228" /> The 3CL Protease Substrate is an internally quenched 14-mer fluorogenic peptide (DABCYL-KTSAVLQSGFRKME-EDANS). When the donor (EDANS) and acceptor (DABCYL) fluorophores are in close proximity the energy emitted from EDANS is quenched by DABCYL (intact substrate). Upon proteolysis by 3CL, the peptide substrate is cleaved between the glutamine and serine residues to generate the highly fluorescent peptide fragment (SGFRKME-EDANS). The fluorescence intensity increases proportionally to the activity of 3CL. More information on the substrate, including MW and

The USP20 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP20 for screening and profiling applications. To determine the effect of an inhibitor on USP20 activity, the enzyme should be preincubated with or without the test inhibitor prior to adding the Ub-AMC substrate to the reaction. The assay was functionally validated using Ub-Aldehyde, a potent inhibitor of DUB subfamilies Ubiquitin C-terminal Hydrolases (UCHs), Ubiquitin-Specific Proteases (USPs), Ovarian Tumor Proteases (OTU), and Machado-Josephin Domain (MJD) proteases. <img src="{{media url="wysiwyg/ubiquitin/78831.png"}}" alt="" width="500" height="226" /> Figure 1: Illustration of the assay principle. <br />Ubiquitin-AMC is a fluorogenic substrate for ubiquitin hydrolases based on the C-terminus derivatization of ubiquitin with 7-amido-4-methylcoumarin (AMC). In the conjugated form, the energy emitted from fluorochrome AMC is quenche

The MDM2-Driven p53 Ubiquitination Assay kit is a sensitive AlphaLisa® high-throughput screening (HTS) assay designed to measure MDM2 (mouse double minute 2 homolog) E3 ligase activity in a homogeneous 384 reaction format. The assay kit comes with enough biotinylated ubiquitin, ATP, FLAG-tagged p53, assay buffer, detection buffer, purified UBE1 (E1), UbcH5b (E2), and MDM2 (E3) for 384 reactions. The assay can detect mono-ubiquitination and poly-ubiquitination of p53. E1 and E2 enzymes are incubated with the E3 complex and FLAG-tagged p53, in the presence of biotin-conjugated ubiquitin and ATP. Ubiquitination of p53 occurs in a multistep ubiquitin transfer from E1 to E2 to E3, and E3-mediated conjugation of ubiquitin to p53. Next, acceptor beads are added, followed by streptavidin-conjugated donor beads. Alpha-counts are then measured. The increase in Alpha-counts is proportional to the mono- or poly-ubiquitination of the FLAG-tagged p53.

The Chemi-Verse™ PAK4 Kinase Assay Kit is designed to measure PAK4 (p21-activated kinase 4) serine/threonine kinase activity for screening and profiling applications using ADP-Glo™ as a detection reagent. The assay kit comes in a convenient 96-well format, with enough purified recombinant PAK4 kinase, kinase substrate, ATP, and kinase assay buffer for 100 enzyme reactions.

The Chemi-Verse™ PAK6 Kinase Assay Kit is designed to measure PAK6 (p21-activated kinase 6) serine/threonine kinase activity for screening and profiling applications using ADP-Glo™ as a detection reagent. The assay kit comes in a convenient 96-well format, with enough purified recombinant PAK6 kinase, kinase substrate, ATP, and kinase assay buffer for 100 enzyme reactions.

The Chemi-Verse™ PAK7 Kinase Assay Kit is designed to measure PAK7 (p21-activated kinase 7) serine/threonine kinase activity for screening and profiling applications using ADP-Glo™ as a detection reagent. The assay kit comes in a convenient 96-well format, with enough purified recombinant PAK7 kinase, kinase substrate, ATP, and kinase assay buffer for 100 enzyme reactions.

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Von Hippel-Lindau, or VHL, is an E3 Ligase that mediates ubiquitination and proteasomal degradation of target proteins. VHL is indicated to play a large role in tumor suppression, particularly due to its interactions with HIF (hypoxia inducible factor). The VHL is typically used as the required E3 ligase in PROTAC (Proteolysis Targeting Chimera) drug design, protein degradation studies and selectivity profiling. VHL complex  is a multi-subunit ubiquitin ligase constituted with VHL, Elongin B, Elongin C, Cullin-2 and Rbx1. Elongin B (ELOB) and Elongin C (ELOC) as a heterodimer binds to the BC-box motif present in SOCS- & VHL-box protein families. VHL is the substrate recognition component that brings target specificity. VHL is linked to scaffolding proteins Cullin-2/Rbx1 of the complex via adaptor protein Elongin C with Elongin B stabilizing the complex.