Functional Assays

The FGL1:LAG3 Inhibitor Screening Assay is an AlphaLISA®-based assay designed to measure the inhibition of LAG3 binding to FGL1 in a homogeneous 384-reaction format. The assay is straightforward: first, FGL1 and LAG3 are incubated with or without a compound of interest. Then, acceptor and donor beads are added to the reaction, followed by reading the Alpha-counts. <img src="{{media url="wysiwyg/Imtx/thumbnail_78824_LAG3-LGL1_illustration.png"}}" alt="" width="900" height="253" /> Illustration of the assay principle.<br />FGL1 contains a His tag recognized by the Nickel chelate acceptor bead, whereas LAG3 is biotinylated, allowing its binding to the streptavidin-coated acceptor bead. Interaction between FGL1 and its receptor LAG3 brings the donor and acceptor beads in proximity. A singlet oxygen generated upon excitation of the donor bead leads to the excitation of the acceptor bead, which emits light. Light emission in the assay is proportional to the level of interaction. AlphaLISA™

The KRAS(G12V) Isoform B Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of antagonists/inhibitors of KRAS(G12V), Isoform B, by monitoring the binding of an effector protein such as the Ras binding domain of Raf1 (RBD-cRaf) to KRAS. First, a sample containing GDP-loaded KRAS(G12V) Isoform B is incubated with SOS1 and GTP for the nucleotide exchange. Next, RBD-cRAF is added and incubated for the effector-RAS binding. Then, acceptor and donor beads are added and incubated for detection followed by reading the Alpha-counts. SOS1 (son of sevenless) is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP. GDP-loaded KRAS(G12V) Isoform B is in an inactive state and does not interact with the Ras-binding domain (RBD) of cRAF.  SOS1 assists in the release of GDP from KRAS(G12V) Isoform B so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS that permits its binding to RBD-cRAF. The KRAS

The Activated Protein C (APC, also known as PROC) Assay Kit is a 96-well format fluorogenic assay designed to measure the protease activity of Activated Protein C (PROC) for screening and profiling applications. <img src="{{media url="wysiwyg/Other/78827_1.png"}}" alt="" width="645" height="251" /> Illustration of the assay principle. <br />The substrate is an internally quenched fluorogenic substrate. Proteolysis releases the highly fluorescent AMC. Fluorescence intensity increases proportionally to the activity of the protease.  

The CSF1R Kinase Assay Kit is designed to measure CSF1R kinase activity for screening and profiling applications using ADP-Glo® as a detection reagent. 

The MMP26 (Matrix metalloproteinase 26) Fluorogenic Assay Kit is designed to measure MMP26 protease activity for screening and profiling applications, in a homogeneous assay with no time-consuming washing steps. 

The USP1 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP1 for screening and profiling applications. To determine the effect of an inhibitor on USP1 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 the DUB subfamilies Ubiquitin C-terminal Hydrolases (UCHs), Ubiquitin-Specific Proteases (USPs), Ovarian Tumor Proteases (OTU), and Machado-Josephin Domain (MJD) proteases.  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 quenched. Upon proteolysis, AMC is no longer quenched and emits fluorescence with an λexcitation/λemission maxima of 350/460 nm. The increase in fluor

The USP5 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP5 for screening and profiling applications. To determine the effect of an inhibitor on USP5 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 the DUB subfamilies Ubiquitin C-terminal Hydrolases (UCHs), Ubiquitin-Specific Proteases (USPs), Ovarian Tumor Proteases (OTU), and Machado-Josephin Domain (MJD) proteases. 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 quenched. Upon proteolysis, AMC is no longer quenched and emits fluorescence with an λexcitation/λemission maxima of 350/460 nm. The increase in fluore

The UCHL1 Inhibitor Screening Assay Kit is a 96-well format fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme UCHL1 for screening and profiling applications. To determine the effect of an inhibitor on UCHL1 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 the 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/Other/78832.png"}}" alt="" width="500" height="205" /> 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 quenched.

The 3CL Protease (T21I, S144A) (SARS-CoV-2) Assay Kit is a 96-well homogeneous fluorogenic assay designed to measure the activity of T21I, S144A mutated 3CL Protease for screening and profiling applications, with no time-consuming washing steps. 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 structure, can be found on our website (BPS Bi