Functional Assays

The rat histone deacetylase 6 (HDAC6) Assay Kit is a 96-well fluorogenic assay designed to measure rat HDAC6 enzymatic activity for screening and profiling applications. In addition, the kit includes a potent HDAC inhibitor, Trichostatin A (TSA), for use as control. The Fluorogenic Rat HDAC6 Assay Kit is based on a unique fluorogenic substrate and developer combination. A fluorescent dye is conjugated to a peptide containing acetyl-lysine. The fluorescence of the conjugated dye is quenched by the peptide. After treatment of the peptide with an HDAC, a developer solution specific for non-acetylated lysine is added to the reaction. If the acetyl group has been removed from the lysine, the developer releases the dye, allowing for fluorescence. In this assay, fluorescence directly correlates with HDAC activity.

The NIMA-related Kinase 7 (NEK7) Kinase Assay Kit is designed to measure NEK7 serine/threonine kinase activity for screening and profiling applications using ADP-Glo™ as a detection reagent. 

The TNF receptor associated factor 6 (TRAF6) TR-FRET Assay Kit is a sensitive TR-FRET Assay Kit, designed to measure TRAF6 (amino acids 51-159) auto-ubiquitination activity in a homogeneous 384 reaction format. This assay measures mono- or poly-ubiquitination. As a homogeneous assay, it requires no time-consuming washing steps and is especially suitable for high throughput screening (HTS) applications as well as real-time analyses. Read out requires a fluorescent plate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The assay takes advantage of biotinylated ubiquitin, which is linked to TRAF6 during auto-ubiquitination. The streptavidin-conjugated acceptor binds to ubiquitin-biotin while a Terbium-conjugated anti-GST antibody donor binds to GST-tagged TRAF6, allowing TR-FRET pairing. The resulting TR-FRET signal is directly proportional to TRAF6 mono or poly-ubiquitination.

The Dicer Fluorogenic Assay Kit is a homogeneous 384-well assay designed to measure the double-stranded-RNA processing activity of Dicer for screening and profiling applications. The Dicer Fluorogenic Assay Kit contains enough purified Dicer, substrate, and assay buffer for 384 reactions. The assay relies on an RNA duplex imitating pre-miRNA, containing a conjugated fluorophore and quencher pair. Fluorescence of the dye is effectively quenched by the proximity of the quencher in the intact RNA duplex. The endoribonuclease Dicer processes the pre-miRNA into a mature 22 bp double-stranded miRNA by cleaving the 5' end which is conjugated to the dye, releasing it from the miRNA. Separated from the quencher, the dye emits fluorescence with excitation/emission maxima of λ=647/ 665 nm. Dicer activity is proportional to the increase in fluorescence.

The Lumi-Verse™ VEGFR2 (KDR) Kinase Assay Kit is designed to measure Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) tyrosine kinase activity for screening and profiling applications, using the highly sensitive ADP-Glo™ as a detection reagent. The assay kit comes in a convenient 96-well format, with enough purified recombinant VEGFR2 (KDR) kinase domain (amino acids 805-1356), kinase substrate, ATP, and kinase assay buffer for 100 enzyme reactions.

The Ubiquitin specific peptidase 2 (USP2) Inhibitor Screening Assay Kit is a homogeneous fluorogenic assay designed to measure the activity of the deubiquitinating (DUB) enzyme USP2 for screening and profiling applications. The kit contains enough purified USP2 recombinant protein, Ubiquitinated-AMC substrate, and assay buffer for 100 reactions. To determine the effect of an inhibitor on USP2 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. 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 q

The MMP7 (Matrix MetalloProteinase 7) Fluorogenic Assay Kit is designed to measure MMP7 protease activity for screening and profiling applications, in a homogeneous assay with no time-consuming washing steps. 

The E. coli in vivo Biotinylation Kit (without BirA Competent Cells) comes in a convenient, easy-to-use format and contains the components necessary to label biotin-acceptor peptides with biotin expressed in E. coli. strain BL21, a chemically competent E. coli B strain. This kit contains sufficient reagents to label 10 x 1 L of E. coli culture. BirA-Transformed chemically competent E. coli cells (BPS Bioscience #27462) are not provided with this kit and must be purchased separately. 

The PARP3 Chemiluminescent Assay Kit is designed to measure PARP3 activity for screening and profiling applications. The PARP3 assay kit 96-well format contains enough purified PARP3 enzyme, histone mixture, activated DNA template, PARP assay buffer and necessary reagents for 100 or 400 enzyme reactions. The key to the PARP3 Chemiluminescent Assay Kit is the biotinylated NAD⁺ and involves 3 reactions. First, histone proteins are coated on a plate. Next, a biotinylated NAD⁺ mix (termed PARP Substrate Mixture) is incubated with the enzyme and an activated DNA template. Finally, streptavidin-HRP is added, followed by ELISA ECL substrate to produce a chemiluminescence signal that can be measured using a chemiluminescence plate reader. *NOTE: As of April 2022, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.