Methyltransferase Assays

MT HotSpot^TM radiometric assay

Methyltransferases use tritium-labeled S-adenosyl-L-methionine (SAM) as the methyl donor that is converted to S-adenosyl-L-homocysteine (SAH) during the transfer of the radioactive methyl group to the histone substrate.

Reaction mixtures are incubated and spotted onto filter papers, which are then washed to remove unreacted SAM, leaving the bound radiolabeled product for detection.

Due to the wash step requirement and the costs and safety concerns associated with the use of radioisotope materials, the radioisotope filter binding assay has traditionally been limited in its application to high-throughput screening. To address these limitations, Reaction Biology has developed the HotSpot^TM assay technology, which is a biologically relevant, nanoliter-scale radioisotope filter binding platform, which offers high-throughput capacity and low material requirements.

The methyltransferase HotSpot^TM assay requires no substrate modification, coupling enzymes, or detection antibodies.

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

Controls: No inhibitor (DMSO vehicle) control and for every assay, one target-specific control compound is tested in 10-dose IC50 format. 

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.

Screening facility: This assay is performed at our screening facility in Malvern, PA, USA.

Compound requirements: In brief, for a standard project, 20 µl of a 10 mM DMSO stock or solid material is needed. Less material is needed for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.

Screening on a panel of methyltransferases is performed on a regular basis every other week.

Please see the upcoming screening dates here.

Screen your own or our libraries for hit identification.

Available 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.

Methyltransferase assays that are not part of our portfolio can be developed in a custom-tailored fashion according to your research needs.

Our assay development service includes:

• Plasmid construction, cDNA synthesis, and mutagenesis
• E. coli or Baculovirus-based expression in insect 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