Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.
The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.
Steinberg
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TH
Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402, USA.
Pretty On Top
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Berggren
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Kemper
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Jones
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Diwu
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Haugland
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Patton
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D000818:Animals; D001360:Avidin / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D005949:Glucose Oxidase / Q000302:isolation & purification; D006001:Glycoconjugates / Q000302:isolation & purification; D006023:Glycoproteins / Q000302:isolation & purification*; D006031:Glycosylation; D007525:Isoelectric Focusing / Q000379:methods; D036103:Nanotechnology; D009961:Orosomucoid / Q000302:isolation & purification; D020543:Proteome / Q000302:isolation & purification
D005456:Fluorescent Dyes; D006001:Glycoconjugates; D006023:Glycoproteins; D009961:Orosomucoid; D020543:Proteome; D001360:Avidin; D005949:Glucose Oxidase