Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis.
The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.
Department of Analytical Chemistry, Faculty of Sciences, Charles University, Prague, Czech Republic. pacakova@natur.cuni.cz
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V
Pacáková
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S
Hubená
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M
Tichá
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M
Mad ra
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K
Stulík
D002021:Buffers; D003959:Diamines; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009961:Orosomucoid / Q000302:isolation & purification; D010047:Ovalbumin / Q000302:isolation & purification; D011107:Polylysine / Q000031:analogs & derivatives*; D020033:Protein Isoforms / Q000302:isolation & purification
D002021:Buffers; D003959:Diamines; D004573:Electrolytes; D006023:Glycoproteins; D009961:Orosomucoid; D020033:Protein Isoforms; C060309:succinyl-polylysine; D011107:Polylysine; D010047:Ovalbumin