Assay Development (La Jolla)

Overview

The Assay Development core facility performs design, development, validation and pilot screening of biological high-throughput assays. Our dedicated staff is skilled in various screening modalities to help design and develop de-novo assays or convert low-throughput lab protocols (e.g. cuvette- or gel-based) into plate-based HTS assays. To date, the facility has developed more than 250 cell-based and biochemical assays. Approximately half of these assays advanced to HTS, and the other half are employed in assay panels supporting HTS hit validation and Structure-Activity Relationship (SAR) studies. The facility is an integral part of Conrad Prebys Center for Chemical Genomics and works closely in collaboration with other cores and teams.

The HTS Assay Development core also provides direct assistance with preparing screening-related grants, guiding through assay development stages, helping design testing funnels, and generating preliminary data.
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Services

  • Consultations on suitable screening alternatives and selection of the approaches consistent with project goals
  • Implementation and optimization of previously established screening protocols
  • Design and development of HTS assays
    • Cell-based and biochemical assays in 96-, 384- and 1536-well plate formats
    • Assays based on variety of HTS detection approaches (absorbance, fluorescence, fluorescence polarization, luminescence, AlphaScreen, BRET, FRET, TR-FRET)
    • Functional and binding assays, including Differential Scanning Fluorimetry (also called protein thermal shift) assays detecting direct small-molecule binding through stabilization of target proteins in thermal denaturation
    • Multiplexed cell-based assays for simultaneous assessment of multiple cellular functions, e.g. multiplexed qPCR assays, or reporter gene expression and cell viability
    • End-point and kinetic assays using common and special screening equipment
  • Assay optimization, miniaturization and implementation
    • Optimization of common assay parameters, such as cell density, ligand and enzyme concentrations, assay media or buffer
    • Evaluation of DMSO concentration effects
    • Optimization of assay sensitivity for identification of compounds with desired MOA
    • Evaluation and optimization of assay statistics and reproducibility
  • Focused library pilot screening
    • Assessment of assay performance measures (signal-to-noise, signal-to-background, assay dynamic range, Z'-factor) and their reproducibility
    • Evaluation and optimization of HTS hit rate
    • Access to a wide selection of focused libraries, e.g. collections of known drugs, kinase inhibitors, NCI compound sets (please inquire about the full list of focused libraries)
  • Support for Structure-Activity Relationship (SAR) studies:
    • Design and implementation of SAR panels relevant to the project goals
    • Development of secondary (e.g. orthogonal, counter-screen) assays
    • SAR data analysis and interpretation
    • Mechanism of action studies: determination of compound's Ki, competition profile and time-dependent effect
  • Assistance with the preparation of screening grants
    • Consultation on available funding mechanisms (e.g. NIH PAR-12-058 and PAR-12-059)
    • Help in development of specific aims and project plan
    • Hands-on support in generation of preliminary data
    • Expertise with diverse target classes and multiple formats utilized at the facility and its application for selecting secondary assays and designing project workflow

Equipment and Resources

Major robotic equipment and resources used by Assay Development at the Conrad Prebys Center for Chemical Genomics include:

  • Two VIIA7, 7th generation real-time PCR instruments
    • Specifically designated for development and screening of qPCR and Differential Scanning Fluorimetry (protein melt) assays
    • 384-, 96-, fast 96-well plate and TaqMan array card assays
  • Hamamatsu FDSS7000 rapid-kinetics (e.g. GPCR, glow luminescence, enzyme kinetics) fluorescence and luminescence plate reader with integrated 384-well liquid handling
  • LabCyte Access Laboratory Workstation for cherry picking of screening hits and preparation of SAR compound plates
  • NEPHELOstar plate reader for detecting light scattering in 96- and 384-well plate based solutions using nephelometry approach
  • Monochromator based multimode plate readers
    • Two Molecular Devices M5 instruments
    • Molecular Devices FlexStation 3
  • Filter based multimode plate readers
    • Molecular Devices Analyst HT equipped with stacker unit
    • Perkin Elmer EnVision equipped with liquid injector unit and 50-plate stackers
  • Microplate pipetting systems for handling serial dilutions and replication of plates for MOA studies
    • Agilent plate processing workstation - includes a Bravo liquid handler, BenchCell 4x with four stackers, a PlateLoc sealer, and Vcode barcode labeler; allows liquid dispensing using full-head and single-row or -column modes applicable for serial diluations
    • BioTek Precision - allows large-volume serial dilutions in 96- and 384-well plate formats and bulk dispense
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