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Related Experiment Videos

A versatile microtiter assay for the universal cdc2 cell cycle regulator.

B Ducommun1, D Beach

  • 1Cold Spring Harbor Laboratory, New York 11724.

Analytical Biochemistry
|May 15, 1990
PubMed
Summary

A new microassay accurately measures p34cdc2 (cell division cycle 2) protein kinase activity. This rapid method utilizes p13suc1 binding for specific detection in various cell types, enabling large-scale sample analysis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • The cell division cycle 2 (cdc2) protein is a key regulator of the cell cycle.
  • Accurate quantification of cdc2 activity is crucial for understanding cell cycle control.
  • Existing methods for measuring cdc2 activity can be time-consuming or lack specificity.

Purpose of the Study:

  • To develop a rapid and specific microassay for quantifying p34cdc2 protein kinase activity.
  • To leverage the high-affinity interaction between p34cdc2 and p13suc1 for assay development.
  • To enable high-throughput analysis of p34cdc2 activity in various biological samples.

Main Methods:

  • Immobilization of purified p13suc1 (from Escherichia coli) onto microtiter plates.
  • Incubation of cellular lysates with immobilized p13suc1 to capture cdc2 and associated proteins.

Related Experiment Videos

  • Assay of captured p34cdc2 activity via histone H1 kinase assay or immunological detection.
  • Optimization of the assay for *Schizosaccharomyces pombe* cell extracts, with applicability to *Xenopus* oocytes and HeLa cells.

    • Main Results:

    • A microassay was successfully developed based on the specific binding of p34cdc2 to immobilized p13suc1.
    • The assay demonstrated the ability to specifically determine p34cdc2 histone H1 kinase activity.
    • The method proved effective for *S. pombe* cell extracts and adaptable to other eukaryotic systems.
    • The assay allows for the determination of kinase activity in a large number of samples efficiently.

    Conclusions:

    • The developed microassay provides a rapid, specific, and high-throughput method for measuring p34cdc2 kinase activity.
    • This assay facilitates the study of cell cycle regulation across different organisms.
    • The p13suc1-based approach offers a valuable tool for biochemical and cell biology research.