HitHunter® Kinase Enzyme Activity Assay Kits
Accelerate the Development of Kinase Inhibitor Therapeutics with Validated, Ready-to-use Kinase Enzyme Activity Assays
Profiling of kinase inhibitors calls for accurate determination of enzyme activity to assess inhibitor selectivity and potency across multiple kinase targets towards the development of safer therapeutics. Relying on a ready-to-use, validated screening platform, such as the HitHunter Kinase Enzyme Activity Assays, offers the high specificity and sensitivity needed to measure specific enzyme activity in a simple user-friendly format. Utilize these assays to screen serine/threonine, tyrosine, and lipid kinases inhibitors. Obtain minimal compound interference. During low to high-throughput screening (HTS) when employing a cleaner target-based detection system that can help accelerate the development of your kinase inhibitor therapeutics.
The HitHunter Kinase Enzyme Activity Assays produce red-shifted fluorescent readouts and demonstrate a wide dynamic range, high specificity, and optimal sensitivity, producing high signal using low amounts of kinase per well. These convenient kits are suitable for 384-, 1536-, and 3456- well plates and include ADP Hunter™ Plus detection reagents. The ADP detection platform is highly tolerant to elevated concentrations of substrate and ATP (up to 1 mM), which is very useful when working with kinases with either high substrate Km or high ATP Km.
Eurofins DiscoverX continues to expand this platform to support your drug discovery efforts. The HitHunter Kinase Enzyme Activity Kits can be leveraged for use with our commercially available extensive portfolio of 456 biologically relevant kinases, including 400 wild type and 56 mutant kinases (primarily active kinases) for characterizing selectivity of inhibitors towards different types of kinases and their activities.
Product Highlights
- Non-radioactive, Homogeneous Assay – No wash, antibody-free kinase assays with a fluorescence gain-of-signal readout allowing for flexible assay read time
- Simple, Rapid Assay Format – User friendly workflow features a 60-minute kinase reaction, followed by a 30-minute incubation with detection reagents
- Highly Specific – Directly measure specific kinase activity avoiding antibody binding to phosphorylation site
- Highly Sensitive – Assays use minimal nanogram amounts of kinase per well resulting in large signal-to-noise ratios and low nanogram IC50 values for inhibitors
The HitHunter Enzyme Activity Assay Kits are ready-to-use, complete kits including the kinase of interest, the kinase substrate, detection reagents, assay buffer, ATP Gold reagent, plates, and detailed reagent preparation protocols. The antibody-free, homogeneous protocol is used for evaluating kinase activity and inhibition directly through the measurement of ADP accumulation. This measurement involves the transfer of a phosphate molecule from the phosphate-donating ATP molecule to its specific substrate.
Key Features
- Wide dynamic range, high sensitivity, and large signal-to-noise ratios
- Low amounts of kinase per well
- Tolerant to high concentrations of substrate and ATP (up to 1 mM), which is useful when working with kinases with either high substrate Km or high ATP Km
- Scalable for 384-, 1536-, or 3456-well plate formats
- Applicable for a wide range of kinases including wild type and mutant kinases
HitHunter Kinase Enzyme Activity Assay Principle
HitHunter Kinase Activity Assay Kits assay principle. The assay principle is based on the ADP Hunter Plus detection method and allows for the measurement of ADP, a universal product of kinase activity. This homogeneous (no wash) assay is non-radioactive with a convenient gain-of-signal assay format and provides results in 90 minutes. This simple two-step assay uses a coupled enzyme reaction system that generates hydrogen peroxide (H2O2) from ADP. Hydrogen peroxide, when combined with ADHP (fluorescent dye precursor) in the presence of peroxidase, generates a fluorescent active resorufin dye. A reaction stop solution is also provided with the kit for added signal and background stabilization to allow for automation.
The HitHunter Enzyme Activity Assay kits are applicable for serine-threonine kinases, tyrosine kinases, and other kinase families.
Kinase Inhibitor Determination
Ready-to-use HitHunter Kinase Enzyme Activity Assay Kits demonstrate compound inhibition of kinase activity across a broad range of kinase families.
Serine/Threonine Protein Kinase Example
COT1 Kinase Enzyme Activity Assay Kit results. The assay was used to quantify ADP accumulation in response to a reference inhibitor and demonstrates a robust and sensitive dose-dependent decrease in ADP levels with an 8.9 signal-to-background ratio and IC50 of 45 nM.
Tyrosine Kinase Example
JAK1 Kinase Enzyme Activity Assay Kit results. The assay was used to quantify ADP accumulation in response to the reference inhibitor and demonstrates a robust and sensitive dose-dependent decrease in ADP levels with a 7.92 signal-to-background ratio and IC50 of 2.97 nM.
Lipid Kinase
PI3 Kinase [p110alpha (H1047R) / p85alpha, active] Enzyme Activity Assay results. The assay (coming soon) was used to quantify ADP accumulation in response to the reference inhibitor and demonstrates a robust and sensitive dose-dependent decrease in ADP levels with a 6.5 signal-to-background ratio and IC50 of 1.9 nM.
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