3.2.3. Synthesis of [32P]-NAD+

The radioactive NAD+ was synthesized from α–[32P]-ATP. The reaction mixture containing 1 mM ATP, 10 MBq of α–[32P]-ATP, 20 mM MgCl2, 2 mM beta-nicotinamide mononucleotide, and 5 mg/mL nicotinamide nucleotide adenylyltransferase 1 in 25 mM Tris-HCl, pH 7.5, was incubated at 37 ◦C for 60 min, then stopped by heating to 80 ◦C for 5 min. The denatured protein was removed by centrifugation.
