HotSpot™ and 33PanQinase™: two radioactive assay formats
Reaction Biology offers two radioactive assay formats that are performed in our screening facilities located in Malvern, PA, USA, and Freiburg, Germany. Both assays are based on the transfer of 33P-labelled phosphate from ATP to the kinase substrate. The methods, however, differ with respect to retention and detection of the phosphorylated substrates. Proteins and peptides in the HotSpot™ assay (performed in the US) are captured via spotting of the reaction mix on a filter membrane. The 33PanQinase™ assay (performed in Germany) is performed with FlashPlate microtiter plates, which are coated with scintillant for detection.
Lipid kinases are screened with ADP-Glo (Promega) at both facilities.
Radiometric assay formats are the gold standard for kinase screening producing reproducible, high-quality data, and directly measure enzyme activity. The catalytic product is directly detected (phosphorylated peptide or protein). False positives and negatives, as caused by indirect formats of detection, are avoided.
Both, the HotSpotTM and 33PanQinaseTM assays allow detection of ATP- as well as substrate competitive and allosteric inhibitors.
HotSpot™ assays are adaptable to any protein or peptide substrate without modification.
Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.
The assay protocol for HotSpot™ Assay is available here.
ATP concentrations: 1 uM, 10 uM or apparent ATP-Km up to 100 uM
Controls: No inhibitor control (DMSO vehicle) and for every kinase, one kinase-specific control compound is tested in 10-dose IC50 format.
Screening facility: Malvern, PA, USA
Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.
Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format.
Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.
Compound requirements: In brief, for a standard project with less than 50 kinases, 20ul of a 10mM DMSO stock or solid material is needed. Please inquire for compound requirements for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.
Setup: Single-dose screening or IC50 value determination with 10 concentrations. Other screening formats are available upon request.
The assay protocol for 33PanQinase™ assay is available here.
ATP concentrations: apparent ATP-Km
Controls: DMSO only control.
Screening facility: Freiburg, Germany
Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to commencement of the studies
Report: A detailed report will be provided including assay conditions, target, and substrate information. The raw data and % enzyme activity will be reported in Excel including IC50 values and curve fitting, if applicable.
Compound requirements: In brief, for a standard project with less than 33 kinases, 100 µl of the stock solution with 100x of the highest assay concentration or solid material. Please inquire for compound requirements for large scale screening.
Library screening: Screen your own or our libraries.
- Drug-like diversified small molecule libraries; > 110,000 compounds
- Focused small libraries including kinase inhibitors, FDA-approved inhibitors, epigenetic inhibitors, natural products, and a clinical collection.
- Fragment libraries including a standard diversified fragment library, Fsp3-enriched fragments library, 3D fragment library, protein-protein interaction fragments, and covalent fragments library.
In addition to our high-throughput primary screening formats, we offer orthogonal screening formats and secondary screening formats. Hits can be characterized for their kinetic, binding affinity, mechanism of action, and intracellular activity.
Our medicinal chemistry partners are available to optimize the structure-activity-relationship of your hits to increase potency and reduce off-target and toxic effects and enhance the pharmacological profile of your lead compounds.
Kinase assays that are not part of our portfolio can be developed in a custom-tailored fashion.
Our assay development service includes:
- Plasmid construction, cDNA synthesis, and mutagenesis
- E. coli or Baculovirus-based expression in Sf9 cells
- Co-expression with co-factors is optional
- Purification via HIS, GST, GST-HIS, HALO-HIS, SUMO-HIS, TXN-HIS or other tags via affinity chromatography
- Tag cleavage and enzyme activation are optional
- Identification of suitable substrate
- Determination of enzyme activity
- Optimization of assay conditions