Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465.
The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 x 10(-4)cm(2) V(-1) s(-1). The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.
Swearingen
Kristian E
KE
Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA.
Dickerson
Jane A
JA
Turner
Emily H
EH
Ramsay
Lauren M
LM
Wojcik
Roza
R
Dovichi
Norman J
NJ
D002919:Chymotrypsinogen / Q000737:chemistry / Q000302:isolation & purification; D016264:Dextran Sulfate / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007768:Lactalbumin / Q000737:chemistry / Q000302:isolation & purification; D007834:Lasers; D010047:Ovalbumin / Q000737:chemistry / Q000302:isolation & purification
D010047:Ovalbumin; D007768:Lactalbumin; D002919:Chymotrypsinogen; D016264:Dextran Sulfate