Separation of histones from Physarum polycephalum by ion-paired, reverse-phase high-performance liquid chromatography

The histones of Physarum polycephalum were efficiently separated using a muBondapak C-18 column with 50 mM triethylamine-phosphoric acid, pH 2.2, as the aqueous phase and a gradient of increasing concentration of acetonitrile. The effects of buffer concentration and changes in gradient rate and flow rate on retention times were investigated to optimize separation. The recovery of total histones (70%) was independent of loading up to at least 0.5 mg. Loading had little effect on retention times. Under optimal conditions, substantially pure (less than 90%) fractions of each histone were obtained. The quantity of each histone after HPLC fractionation reflected its abundance in the original sample, indicating that there was no selective loss of a particular histone.