Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

J Chromatogr A. 2008 Jun 20;1194(2):249-52. doi: 10.1016/j.chroma.2008.04.047. Epub 2008 Apr 24.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chymotrypsinogen / chemistry
  • Chymotrypsinogen / isolation & purification
  • Dextran Sulfate / chemistry
  • Electrophoresis, Capillary / methods*
  • Fluorescence*
  • Lactalbumin / chemistry
  • Lactalbumin / isolation & purification
  • Lasers*
  • Ovalbumin / chemistry
  • Ovalbumin / isolation & purification

Substances

  • Ovalbumin
  • Lactalbumin
  • Chymotrypsinogen
  • Dextran Sulfate