High-resolution capillary isoelectric focusing of complex protein mixturesfrom lysates of microorganisms

High-resolution capillary isoelectric focusing separations of complex prote in mixtures have been obtained for cellular lysates of Saccharomyces cerevi siae, Eschericia coli, and Deinococcus radiodurans, High quality separation s are shown to be achievable for total protein concentrations of <0.1 mg/mL . The separation reproducibility was examined, and the influence of the cap illary inner wall coating on resolution investigated using fused-silica cap illaries coated with various hydrophilic polymers including hydroxypropyl c ellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having a n isoelectric point (pI) difference of 0.004 are shown to be separated usin g a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pi range of 5-7 were obtained for S. cereuisiae, similar to 80 peaks in the pi range of 4.5-8.5 for E. c oil, and similar to 210 peaks in the pi range of 3-8.8 for D. radiodurans.