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A microtiter plate assay for protein kinase C
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Analytical Biochemistry
|June 1, 1987
Summary
A new microtiter plate assay for protein kinase C (PKC) offers a faster method for enzyme activity measurement. This assay provides reliable results, making it ideal for screening chromatography fractions during purification.
Area of Science:
- Biochemistry
- Enzymology
- Assay Development
Background:
- Protein kinase C (PKC) plays a crucial role in cellular signaling pathways.
- Accurate and efficient assays are essential for studying PKC activity, particularly during protein purification.
- Traditional assays can be time-consuming and labor-intensive.
Purpose of the Study:
- To develop a novel microtiter plate-based assay for quantifying protein kinase C activity.
- To establish a rapid and reliable method for assessing enzyme activity in various samples.
Main Methods:
- A 96-well microtiter plate format was utilized for the protein kinase C assay.
- Enzyme samples and reaction components were incubated with [gamma-32P]ATP.
- Radioactivity was measured after spotting samples onto phosphocellulose filter paper and washing.
Main Results:
- The microtiter plate assay demonstrated comparable results to standard tube-based assays.
- The assay exhibited high reproducibility with intra-assay variation <9% and inter-assay variation <5%.
- Assay linearity was observed with incubation time up to 20 minutes and varying enzyme concentrations.
Conclusions:
- The developed microtiter plate assay is a rapid and efficient method for protein kinase C activity determination.
- This assay is suitable for high-throughput screening of column chromatography fractions.
- The method can be adapted for assaying other kinase activities.