COMPARISON OF AMPHOLYTES USED FOR SLAB GEL AND CAPILLARY ISOELECTRIC-FOCUSING OF RECOMBINANT TISSUE-TYPE PLASMINOGEN-ACTIVATOR GLYCOFORMS

Four commercial ampholytes: Ampholine and Pharmalyte (Pharmacia Biotec h), Bio-Lyte (Bio-Rad) and Servalyt (Serva) were evaluated for their a bility to resolve recombinant tissue-type plasminogen activator (rt-PA ) glycoforms by isoelectric focusing (IEF) and capillary IEF (cIEF). E ach brand of ampholytes focused rt-PA into 3-4 major and 5-6 minor ban ds on slab gel electrophoresis. Visually, focused bands stained with C oomassie Blue appeared to be similarly resolved by all the ampholytes except for Ampholines, where the bands were closely grouped and more i ntensely stained. When cIEF was performed, Pharmalytes and Ampholines resolved rt-PA glycoforms consistent with the slab gels. No discernibl e peaks were detected during cIEF of rt-PA using Servalyts or Bio-Lyte s. UV spectrophotometric scans of the components used for cIEF showed that Servalyts absorbed intensely over a range which overlapped the de tector bandpass. Bio-Lytes showed absorption over a narrower UV range but still overlapped the detector bandpass, thus preventing the discer nment of protein peaks. For this cIEF system the best ampholytes were Ampholines and Pharmalytes.