Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [γ-32P]ATP Analog from ADP Analog
This protocol was adapted from “Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates,” Chapter 24, in Protein-Protein Interactions (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
The generation of a high-specific-activity [γ-32P]ATP analog is essential for detecting direct substrates of a kinase. Synthesis of radiolabeled ATP analog from unlabeled ADP analog involves a succession of phosphotransfer reactions that are performed in two stages. The first stage involves transfer of phosphate from [γ-32P]ATP to nucleotide diphosphate kinase (NDPK) to make [32P]NDPK. The second stage involves transfer of labeled phosphate from [32P]NDPK to ADP analog to generate [γ-32P]ATP analog. In this protocol, radiolabeled [γ-32P]cpATP is prepared from cyclopentyl ADP (cpADP) and [γ-32P]ATP by a phosphate transfer reaction using NDPK.
