Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [{gamma}-32P]ATP...

INTRODUCTIONThe generation of a high-specific-activity [γ-(32)P]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-(32)P]ATP to nucleotide diphosphate kinase (NDPK) to make [(32)P]NDPK. The second stage involves transfer of labeled phosphate from [(32)P]NDPK to ADP analog to generate [γ-(32)P]ATP analog. In this protocol, radiolabeled [γ-(32)P]cpATP is prepared from cyclopentyl ADP (cpADP) and [γ-(32)P]ATP by a phosphate transfer reaction using NDPK.