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Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection.
Electrophoresis. 2006 Apr; 27(7):1347-54.E

Abstract

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.

Authors+Show Affiliations

Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16568403

Citation

Yan, Weiying, et al. "Protein Labeling With Red Squarylium Dyes for Analysis By Capillary Electrophoresis With Laser-induced Fluorescence Detection." Electrophoresis, vol. 27, no. 7, 2006, pp. 1347-54.
Yan W, Sloat AL, Yagi S, et al. Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis. 2006;27(7):1347-54.
Yan, W., Sloat, A. L., Yagi, S., Nakazumi, H., & Colyer, C. L. (2006). Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis, 27(7), 1347-54.
Yan W, et al. Protein Labeling With Red Squarylium Dyes for Analysis By Capillary Electrophoresis With Laser-induced Fluorescence Detection. Electrophoresis. 2006;27(7):1347-54. PubMed PMID: 16568403.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection. AU - Yan,Weiying, AU - Sloat,Amy L, AU - Yagi,Shigeyuki, AU - Nakazumi,Hiroyuki, AU - Colyer,Christa L, PY - 2006/3/29/pubmed PY - 2006/7/4/medline PY - 2006/3/29/entrez SP - 1347 EP - 54 JF - Electrophoresis JO - Electrophoresis VL - 27 IS - 7 N2 - Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/16568403/Protein_labeling_with_red_squarylium_dyes_for_analysis_by_capillary_electrophoresis_with_laser_induced_fluorescence_detection_ DB - PRIME DP - Unbound Medicine ER -