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Related Experiment Videos

Nonradioactive assay for sequence-specific DNA binding proteins

M L Gubler1, P Abarzúa

  • 1Department of Oncology, Roche Research Center, Hoffmann-LaRoche Inc., Nutley, NJ 07110, USA.

Biotechniques
|June 1, 1995
PubMed
Summary

A novel nonradioactive assay quantitates DNA binding proteins using 96-well microplates for high throughput. This functional assay accurately measures sequence-specific DNA binding and correlates with biological function.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Assay Development

Background:

  • Quantifying DNA binding proteins is crucial for understanding gene regulation.
  • Existing methods can be time-consuming or require radioactivity.
  • High-throughput assays are needed for large-scale screening.

Purpose of the Study:

  • To develop a novel, nonradioactive functional assay for quantifying DNA binding proteins.
  • To enable high-throughput analysis using 96-well microplates.
  • To validate the assay's ability to measure sequence-specific DNA binding and functional correlation.

Main Methods:

  • Development of a nonradioactive assay for DNA binding proteins.
  • Utilization of 96-well microplate format for high sample throughput.

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  • Testing with purified proteins and whole cell extracts.
  • Validation using a temperature-sensitive DNA binding protein.

    • Main Results:

    • The assay successfully quantitates DNA binding proteins in a nonradioactive manner.
    • High-throughput analysis is achievable due to the 96-well microplate format.
    • The assay measures sequence-specific DNA binding accurately in both purified proteins and cell extracts.
    • Demonstrated correlation between measured DNA binding and biological function.

    Conclusions:

    • A novel, nonradioactive, high-throughput assay for DNA binding proteins has been established.
    • This assay provides a reliable method for measuring sequence-specific DNA binding.
    • The assay's ability to correlate with biological function makes it valuable for various research applications.