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Related Experiment Videos

Peptide protein binding assay using ImageFlashPlates or Imaging Beads.

Susanne E Merk1, Hans-Dieter Schubert, Werner Moreth

  • 1Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str.65, D-88397 Biberach, Germany.

Combinatorial Chemistry & High Throughput Screening
|December 8, 2004
PubMed
Summary

This study compares ImageFlashPlates and Imaging Beads for peptide-protein binding assays. Beads offer better assay performance (higher Z

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Area of Science:

  • Biochemistry
  • Assay Development
  • Radioligand Binding Assays

Background:

  • Charge-coupled device (CCD) cameras are standard for radioligand-receptor binding assays, favoring red-shifted scintillation.
  • Historically, red-shifted scintillants were limited to scintillation proximity assay (SPA) Imaging Beads.
  • ImageFlashPlates now offer red-shifted light emission at 615 nm upon beta-radiation exposure.

Purpose of the Study:

  • To establish peptide-protein binding assays using streptavidin-coated ImageFlashPlates and Imaging Beads.
  • To evaluate the performance of these formats in a low volume 384-well format for high-throughput screening (HTS).
  • To compare assay efficiency, signal strength, and statistical performance (Z' value) between the two formats.

Main Methods:

  • Peptide-protein binding assays were conducted using a biotinylated peptide X and [33P]-phosphorylated protein Y.

Related Experiment Videos

  • Assays were performed in low volume 384-well plates using either ImageFlashPlates or Imaging Beads.
  • Both formats underwent specific preparation steps: washing for FlashPlates and centrifugation for beads, prior to scintillation measurement.

    • Main Results:

    • Both ImageFlashPlates and Imaging Beads demonstrated saturable binding of [33P]-phosphorylated protein Y with comparable scintillation efficiency.
    • The bead-based assay yielded a KD value of approximately 30 nmol/l.
    • ImageFlashPlates showed a lower Z' value (0.64) compared to Imaging Beads (0.87) due to signal loss during the washing step, while competition assays yielded similar IC50 values.

    Conclusions:

    • Both ImageFlashPlates and Imaging Beads are suitable for low volume 384-well peptide-protein binding assays applicable to HTS.
    • Imaging Beads demonstrated superior assay performance with a higher Z' value compared to ImageFlashPlates.
    • The choice between formats may depend on automation capabilities, considering the washing step for FlashPlates versus centrifugation for beads.