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Related Experiment Videos

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format.

A R Asthagiri1, A F Horwitz, D A Lauffenburger

  • 1Department of Chemical Engineering and Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Analytical Biochemistry
|May 1, 1999
PubMed
Summary

We developed a sensitive and quantitative in vitro kinase activity assay for studying protein kinases. This improved method enhances throughput and reduces waste, aiding research into cell signaling and behavior.

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Area of Science:

  • Cellular signaling pathways
  • Molecular biology techniques
  • Biochemistry

Background:

  • Protein kinases regulate critical cell functions like differentiation and proliferation.
  • Accurate measurement of kinase activity is vital for understanding cell signaling.
  • Traditional assays are often cumbersome and generate significant waste.

Purpose of the Study:

  • To develop a sensitive, quantitative, and high-throughput in vitro assay for measuring protein kinase activity.
  • To improve upon existing kinase assay methodologies, reducing sample requirements and waste generation.
  • To provide a versatile assay applicable to various protein kinases.

Main Methods:

  • A modified in vitro kinase activity assay utilizing antibody immobilization on microtiter wells.

Related Experiment Videos

  • Immunoseparation technique replacing traditional immunoprecipitation.
  • 96-well plate and filter plate configuration for increased throughput and reduced radioactive waste.
  • Case study measuring extracellular signal-regulated kinase 2 (ERK2) activity.

    • Main Results:

    • The assay demonstrates high sensitivity, requiring an order of magnitude less cell lysate.
    • The immobilized antibody method simplifies sample handling compared to immunoprecipitation.
    • The 96-well format facilitates high-throughput analysis and reduces radioactive liquid waste.
    • Successful application in measuring ERK2 activity.

    Conclusions:

    • The developed assay is a sensitive, quantitative, and efficient tool for studying protein kinase activity.
    • This method offers significant advantages over traditional assays, including reduced sample input and waste.
    • The assay is adaptable for studying a wide range of protein kinases relevant to cell signaling research.