HTRF® Enzyme Assays

Formed and disrupted HTRF® complex.Lipid Kinase Assays
The HTRF® Lipid Kinase Assay is a high-performance assay kit that provides a universal method for assaying all Class I PI3-Kinases, Class II PI3-Kinases, and PIPKINS in a homogeneous 384- or 1536-well format.
Product Listing
Assay Highlights
  • Large Stokes shift and time-resolved signal reduce compound interference, minimizing false negatives
  • Picomole sensitivity enables the integrity of enzyme kinetics to be retained, ensuring the most physiologically relevant assay parameters
  • 4-step or 3-step assay configurations, to optimise assay volume and throughput for all screening requirements. Read our Kit Selection Guide.
  • Reliable, proven assay—based on the technology used in the Millipore, Upstate® KinaseProfiler™ Service
  • Z` factor exceeding 0.8, ensuring consistent, reliable data
Titration of PI103 against PI3-kinase performance.
The HTRF® Assay Format
Our industry-leading PI3-Kinase assay makes use of the specific, high affinity binding of the GRP1 pleckstrin homology (PH) domain to PIP3, the product of a Class I PI 3-Kinase acting on its physiological substrate PIP2. During the detection phase of the assay, a complex is generated between the GST-tagged PH domain and biotinylated short chain PIP3. The biotinylated PIP3 and the GST-tagged PH domain recruit fluorophores (Streptavidin-Allophycocyanin and Europium-labelled anti-GST, respectively) to form the fluorescence resonance energy transfer (FRET) architecture, generating a stable time-resolved FRET signal. The FRET complex can be disrupted in a competitive manner by non-biotinylated PIP3, a product formed in the PI 3-Kinase assay.

The Class II PI3K assays are similarly designed, with different lipid-binding domains replacing the GRP1 as required in order to interact with the different lipids involved.

Graph showing the inhibition of PI3-kinase isoforms.