Characterization of each isoform of a F(ab')2 by capillary electrophoresis.
Free solution capillary electrophoresis was investigated for the characterization of an M(r) 100 000 purified F(ab')2. Optimization of the experimental conditions allowed the identification of five separated peaks, suggesting the presence of isoforms which differed by only 0.2 pH unit. This heterogeneity was still detectable with 80 amol of protein. After a preparative separation by chromatofocusing, identification of each form was performed for the first time by capillary electrophoresis. A quantitative and qualitative correlation with isoelectric focusing showed that free solution capillary electrophoresis represents a sensitive method for revealing subtle differences in charge, even for large proteins.
Vincentelli
R
R
T.M. Innovation (Centre National de Transfusion Sanguine), Institut Merieux, France.
Bihoreau
N
N
D004586:Electrophoresis / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D007525:Isoelectric Focusing; D010434:Pepsin A; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification*
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D020033:Protein Isoforms; D010434:Pepsin A