Reaction of fluorogenic reagents with proteins III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503.
The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were determined by continually infusing Chromeo P503-labeled alpha-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concentration than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis analysis of Chromeo P503-labeled alpha-lactalbumin produced concentration detection limits (3sigma) of 12 pM and mass detection limits of 0.7 zmol, but with modest theoretical plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis analysis of a homogenate prepared from a Barrett's esophagus cell line; the separation quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.
Turner
Emily H
EH
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Dickerson
Jane A
JA
Ramsay
Lauren M
LM
Swearingen
Kristian E
KE
Wojcik
Roza
R
Dovichi
Norman J
NJ
D002460:Cell Line; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry; D007834:Lasers; D038761:Photobleaching; D011166:Porphyrins / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
D011166:Porphyrins; D011506:Proteins; C006101:P503; D007768:Lactalbumin