Inositol Phosphate Kinase Assay

The reaction mix contained: 2 µl 5× Buffer (150 mM Hepes 6.8; 250 mM NaCl; 30 mM MgSO₄; 5 mM DTT; 5 mM NaF); 0.5 µl phosphocreatine (200 mM); 0.5 µl creatine phosphokinase (800 U/µl) ; 0.5 µl ATP-Mg (10 mM) ; 2–10 nmol IP₅/IP₆/IP₇; and 5 to 30 ng of the appropriate enzyme. Trace amounts of [³H]-IP₅ or [³H]-IP₆ (∼20,000 CPMs) was added when indicated. The reactions were incubated at 37°C for the indicated times. Reactions were then stopped by the addition of 2 µl EDTA (100 mM) and placed on ice. The samples were then run on a polyacrylamide gel, frozen at −20°C, or processed for SAX-HPLC analysis as previously described [25] (link). Briefly, 50 µl of 1 M perchloric acid was added to the samples followed by the addition of 25–30 µl of 1 M potassium carbonate containing 3 mM EDTA to neutralize the mixture.