High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms.
High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using 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. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.
Shen
Y
Y
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Xiang
F
F
Veenstra
T D
TD
Fung
E N
EN
Smith
R D
RD
D001426:Bacterial Proteins / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D005656:Fungal Proteins / Q000302:isolation & purification*; D006095:Gram-Positive Cocci / Q000737:chemistry; D007525:Isoelectric Focusing / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D001426:Bacterial Proteins; D005656:Fungal Proteins