Isoelectric focusing by free solution capillary electrophoresis.
A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pI's) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pI's determined in the same experiment. The pI's of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.
Chen
S M
SM
Applied Biosystems, Inc., Lincoln Centre Drive, Foster City, California 94404.
Wiktorowicz
J E
JE
D001426:Bacterial Proteins; D002256:Carbonic Anhydrases / Q000302:isolation & purification; D002766:Cholecystokinin / Q000302:isolation & purification; D004355:Drug Stability; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D007782:Lactoglobulins / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results; D006163:Ribonuclease T1 / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification
D001426:Bacterial Proteins; D007782:Lactoglobulins; D010446:Peptide Fragments; D011506:Proteins; C047902:cholecystokinin C-terminal flanking peptide; D002766:Cholecystokinin; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D002256:Carbonic Anhydrases