Functional Assays

ChoosE2-Opti™ Intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to identify E2 enzyme(s) that are functional partners for a purified E3 ubiquitin ligase of interest and lead to its self-polyubiquitination. The assay kit contains five E2 enzymes to be tested with the E3 ligase of interest, and two E3 ligases (SMURF1 and MDM2) to use as internal controls.  It also contains an E1 enzyme, ATP, an optimized TRF Ubiquitin Mix, and a universal buffer.  It utilizes a Europium-labeled Ubiquitin Donor as well as Cy5-labeled Ubiquitin Acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into polyubiquitin chains, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses.

The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12C) antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS(G12C). With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS(G12C) 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(G12C) 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(G12C) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change

The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12D) antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS(G12D). With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS(G12D) 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(G12D) 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(G12D) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change

Please note this product may be subject to fees, we invite you to contact your local office. The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS IsoformB antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS. With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS Isoform B is incubated with SOS1 and GTP for the nucleotide exchange. Next, RBD-cRAF is added and incubated for the <br />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 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

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

The full length PARP15 (PARP15-FL) Chemiluminescent Assay Kit is designed to measure PARP15 activity for screening and profiling applications. PARP15 is known to catalyze the NAD-dependent ADP-ribosylation of histones. This Assay Kit takes advantage of the sensitive detection of biotinylated NAD+. First, histone proteins are coated on a 96-well plate. Next, a biotinylated NAD+ mix (termed PARP Substrate Mixture) is incubated with the PARP15-FL enzyme in an optimized assay buffer. Finally, the plate is treated with streptavidin-HRP followed by addition of the ELISA ECL substrate to produce chemiluminescence that can be measured using a chemiluminescence reader. <img src="{{media url="wysiwyg/parp15pic.jpg"}}" alt="" width="825" height="244" /> Illustration 1: PARP15-FL Chemiluminescent Assay Kit schematic

The TRAF6 intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure TRAF6 auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes a Europium-labeled ubiquitin (Ub) donor as well as Cy5-labeled Ub acceptor to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on TRAF6, this assay measures poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time analyses.

The Induced myeloid leukemia cell differentiation protein (MCL-1) TR-FRET Assay Kit (Rat) is designed to measure the inhibition of rat MCL-1 binding to its ligand in a homogeneous 384-well format. This TR-FRET based assay requires no time-consuming washing steps, making it especially suitable for high throughput screening applications. The assay is straightforward and fast. The TR-FRET signal is measured after a 1-hour incubation of the anti-His terbium (Tb)-labeled donor, dye-labeled acceptor, recombinant Rat MCL-1 protein, peptide ligand, and the inhibitor of interest.

This product is a component in Lifsensors’ UC101: E3 Lite Customizable Ubiquiin Ligase Kit.