Comparison of inositol pyrophosphate recovery between SAX-HPLC and PAGE technology.

Parallel kinase reactions containing IP6K1 and 2 nmols of IP6 or 2 nmol of IP5 and trace amounts of 3H-IP6 or 3H-IP5 were run for two hours at 37°C. One set of reaction products were analyzed by SAX-HPLC after incubating the samples for 20 minutes with percloric acid (1 M) on ice to precipitate the proteins. The second set of reactions were resolved by PAGE and subjected to DAPI staining, the bands corresponding to inositol polyphosphates were then cut and subjected to scintillation counting. The data (CPM) represent the averages+/−standard deviation of three independent experiments. The percent values represent the proportional ratio of the inositol polyphosphates species recovered.