Protocol

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [-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 [-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 [-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 [-³²P]ATP analog. In this protocol, radiolabeled [-³²P]cpATP is prepared from cyclopentyl ADP (cpADP) and [-³²P]ATP by a phosphate transfer reaction using NDPK.