Biochemical Assay Development

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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:

  • Kinases
  • Proteases
  • 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.

Isothermal Titration Calorimetry
iochemical Assay Development_graph 2

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.

Biochemical Assay Development_graph 3
Biochemical Assay Development_graph 4.docx

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.

Assay Development