Protease Assays

Our protease assay panel provides a platform for profiling and screening of new protease inhibitors against all major classes of proteases, such as serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases. Selective compounds can be further characterized via both biochemical and biophysical platforms, such as surface plasmon resonance for their kinetic profile and mechanism of action.

Reaction Biology's protease assays are based on the cleavage of fluorogenic peptide substrates for quantification of the proteolytic activity of proteases enabling the screening of direct and allosteric inhibitors.

Proteases are targeted successfully for the treatment of virus infections such as HIV and Hepatitis C. Inhibitors have been approved for the treatment of hypertension (ACE inhibitors), Diabetes (DPP-IV inhibitor), and to prevent blood clots (Factor Xa inhibitors). Studies are also underway for the treatment of Alzheimer's, COVID-19, and cancer. 

  • Fluorescence-based protease activity assay
  • Low and large scale protease screening, as well as high-throughput screening options, are available
  • Full protease panel protease profiling is performed on a monthly basis
  • Discover our new COVID-19-related protease assays here

Protease Assay Details

Assay Format, 5-FAM

principle of protease assays using fluorescent peptide for compound screening

 Assay procedure for MMP1. Internally quenched fluorescent peptide substrates contain a fluorophore (5-FAM) and a quencher (QXL 520). A fluorescence signal is generated upon peptide cleavage which is quantified for determination of protease activity.

Assay Setup

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, US.

Compound requirements: In brief, for a standard project, 20ul of a 10mM 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.

Monthly selectivity screening

On a monthly basis, Reaction Biology performs compound screening on the full protease panel.

Please see the upcoming screening dates here.