El. Gump et Ca. Monnig, PRECOLUMN DERIVATIZATION OF PROTEINS TO ENHANCE DETECTION SENSITIVITYFOR SODIUM DODECYL-SULFATE NON-GEL SIEVING CAPILLARY ELECTROPHORESIS, Journal of chromatography, 715(1), 1995, pp. 167-177
Citations number
36
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
Pre-column derivatization of proteins with fluorescamine, naphthalene-
2,3-dicarboxyaldehyde, and o-phthaldialdehyde was used to enhance abso
rption and fluorescence detection after separation by SDS non-gel siev
ing capillary electrophoresis. When compared with underivatized protei
ns, absorption sensitivity increased by as much as a factor of 22 at 2
80 nm, and 1.74 and 4.71 at 200 and 220 nm, respectively. Under favora
ble conditions, absorption detection limits with the labeled proteins
at 280 Mn were approximately equivalent to the detection limits of und
erivatized proteins at 200 nm. Fluorescence detection provided attomol
e detection limits with the best results being obtained with high-mole
cular-mass proteins. Pre-column labeling decreased the efficiency of t
he separation, but did not give rise to multiple peaks from heterogene
ous labeling. The migration velocity of labeled proteins was slightly
different from the unlabeled molecules, but did not significantly degr
ade molecular mass determinations. Pre-column derivatization with fluo
rescence detection allowed the proteins in a fertilization membrane is
olated from a single amphibian embryo to be easily characterized.