Using genetically engineered kinases to screen for novel protein kinase substrates: generation of [γ-³²P]ATP analog from ADP analog

Abstract

The generation of a high-specific-activity [γ³²P]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 [γ³²P]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 [γ³²P]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.