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Related Concept Videos

Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...

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Permeabilization strategies to study protein phosphorylation.

A N Carter1

  • 1The Salk Institute for Biological Studies, La Jolla, California, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary
This summary is machine-generated.

This study details methods for labeling proteins using nucleotide triphosphates, specifically [gamma-32P]ATP, in cellular fractions and permeabilized cells. It provides protocols for protein phosphorylation analysis and kinase activity assessment.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Protein phosphorylation is a key regulatory mechanism in cellular processes.
  • Accurate methods are needed to study protein phosphorylation and kinase activity.

Purpose of the Study:

  • To describe in vitro methods for labeling proteins using nucleotide triphosphates.
  • To provide protocols for analyzing protein phosphorylation in permeabilized cells and isolated organelles.
  • To outline techniques for direct analysis of kinase activity.

Main Methods:

  • Utilizing [gamma-32P]ATP or [gamma-32P]GTP as phosphate donors for protein labeling.
  • Performing protein phosphorylation experiments in permeabilized cells and isolated intracellular organelles.
  • Employing immunoprecipitation and electrophoretic analysis for protein identification.
  • Describing methods for direct analysis of cytosolic and membrane-bound kinases.
  • Including procedures for determining the specific radioactivity of 32P-labeled compounds.

Main Results:

  • Established protocols for in vitro protein labeling and phosphorylation analysis.
  • Demonstrated immunoprecipitation of phosphorylated proteins from cell lysates.
  • Presented alternative techniques for lysate preparation and direct kinase analysis.
  • Provided methods for quantifying radioactivity in 32P-labeled molecules.

Conclusions:

  • The described methods enable robust study of protein phosphorylation and kinase activity.
  • These techniques are valuable for investigating cellular signaling pathways.
  • The protocols offer flexibility for analyzing various cellular fractions and kinase types.