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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [{gamma}-32P]ATP
CSH Protocols
|March 2, 2011
Summary
This study details a two-stage method for synthesizing high-specific-activity radiolabeled ATP analogs, crucial for kinase substrate detection. The protocol efficiently produces [γ-(32)P]cyclopentyl ATP ([γ-(32)P]cpATP) using nucleotide diphosphate kinase.
Area of Science:
- Biochemistry
- Molecular Biology
- Radiochemistry
Background:
- High-specific-activity radiolabeled adenosine triphosphate (ATP) analogs are vital for identifying direct kinase substrates.
- Current synthesis methods involve multi-step phosphotransfer reactions.
Purpose of the Study:
- To present a streamlined protocol for generating a high-specific-activity [γ-(32)P]ATP analog, specifically [γ-(32)P]cyclopentyl ATP ([γ-(32)P]cpATP).
- To enable efficient detection of direct kinase substrates.
Main Methods:
- The synthesis employs a two-stage phosphotransfer reaction catalyzed by nucleotide diphosphate kinase (NDPK).
- Stage 1: Phosphorylation of NDPK using [γ-(32)P]ATP to form [(32)P]NDPK.
- Stage 2: Transfer of the radiolabeled phosphate from [(32)P]NDPK to cyclopentyl ADP (cpADP) to yield [γ-(32)P]cpATP.
Main Results:
- Successfully synthesized high-specific-activity [γ-(32)P]cpATP.
- The protocol utilizes readily available reagents and established enzymatic reactions.
Conclusions:
- The described method provides an efficient and reliable means for preparing [γ-(32)P]cpATP.
- This radiolabeled analog is a valuable tool for kinase research and substrate identification.
