Biochemical assay development is often used as the primary assay in a drug discovery screening cascade due to a combination of their accessibility (availability of recombinant protein), scalability, throughput and robustness.
The primary assay for the target of interest is often followed by a selectivity assay for closely related family members of the target, often using a similar assay technology.
Our scientists have a broad experience in assay development, validating and using biochemical assays for a range of enzyme targets, protein-protein interaction studies and binding assays.
Target classes include, but are not limited to:
- Histone deacetylases (HDAC)
- Methyl transferases
We aim to develop assays in a 384 well format to maximize throughput and minimize reagent consumption. When developing a biochemical assay, we will optimize the assay buffer and co-factors, determine the substrate Km, optimal enzyme concentration and suitable incubation time to maximize assay sensitivity.
Obtaining Optimal Results for Biochemical Assay Development
We have a suite of multi-modal plate readers that allow us to utilise the full range of relevant technologies. These include FRET, BRET, fluorescence intensity, absorbance, luminescence, AlphaLISA, Time-Resolved Fluorescence (TRF) and Fluorescence Polarization (FP).
Our BMG Clariostar plate readers are equipped with temperature and gaseous control, enabling kinetic measurements at a defined temperature. We have filter based and monochromator readers (Revivity and BMG) allowing for optimization of appropriate excitation and emission for fluorescent probes.
When completing a biochemical assay development we will select, where possible, suitable fluorescent probes that minimize the potential for compound interference and resulting in reduced false positives.
Fig 1. Binding assay using Fluorescence Polarisation (FP) e.g. Bodipy cyclopamine 1ug of Smoothened membrane per well (12mg/ml stock), 3nM Bodipy-Cyclopamine, assay volume 15ul, Buffer conditions: 25mM HEPES pH 7.4, 2mM EDTA, 0.05% bovine γ globulin, 0.01% pluronic acid, protease inhibitors.
Fig 2. Initial rate determination of enzyme turnover at varying substrate concentrations then transformed into a Km determination plot
Fig 3. Dose response curves to standard inhibitors of neutrophil elastase. The assay utilised the fluorescent substrate, MeOSuc-AAPV-AMC.