Application of capillary isoelectric focusing with universal concentration gradient detector to the analysis of protein samples.
The design of a new capillary isoelectric focusing (cIEF) instrument, composed of a rugged cartridge holding a short piece of capillary and a universal, inexpensive concentration gradient detector, was optimized and applied to the analysis of various protein samples. High-efficiency cIEF separations with sub-femtomole detection limits for absolute amounts were obtained using 10 microns I.D. capillaries with large O.D.-to-I.D. ratios. An electric field strength of 1 kV/cm applied in the focusing step resulted in a 10(-8) M on-column concentration detection limit, which corresponded to 10(2) amol absolute amount of proteins. The detection volume was estimated to be 2 pl, which is among the smallest values reported to date for any optical or spectroscopic detector. When a 6-cm long capillary was used, proteins with isoelectric points ranging from 4.7 to 8.8 could be analyzed in about 5 min, the shortest analysis time ever reported for cIEF. Compared with commercial cIEF instruments with UV-visible absorbance detectors, the instrument is easier to use and has lower detection limits and better resolution. Several protein mixtures and real samples were separated with this instrument.
Wu
J
J
Department of Chemistry, University of Waterloo, Ontario, Canada.
Pawliszyn
J
J
D000911:Antibodies, Monoclonal / Q000032:analysis; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007834:Lasers; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000032:analysis
D000911:Antibodies, Monoclonal; D006454:Hemoglobins; D007202:Indicators and Reagents; D011506:Proteins; D012709:Serum Albumin; D014168:Transferrin