Micellar electrokinetic chromatography with diode laser-induced fluorescence detection as a tool for investigating the fluorescence labeling of proteins.
We describe the use of micellar electrokinetic chromatography with diode laser-induced fluorometry (MEKC-DLIF) as a tool to characterize a labeling reaction between sulfoindocyanine succinimidyl ester (Cy5), a cyanine fluorescent dye, and a model protein, bovine serum albumin (BSA). To decrease the influence of imprecise injection, methylene blue was added as an internal standard. Labeled BSA was completely separated from the unconjugated Cy5, and methylene blue under optimized conditions. A kinetic study of the reaction was performed by changing some parameters, such as reaction buffer pH, reaction buffer concentration and the initial concentration of BSA. A comparison between the current method and traditional methods was made.
Jing
Peng
P
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka, Japan.
Kaneta
Takashi
T
Imasaka
Totaro
T
D000818:Animals; D002232:Carbocyanines / Q000032:analysis / Q000737:chemistry; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D007700:Kinetics; D007834:Lasers; D008751:Methylene Blue; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; C085321:cyanine dye 5; D012710:Serum Albumin, Bovine; D008751:Methylene Blue