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 glyc oproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. T he dye then reacts with the aldehydes to generate a highly fluorescent conj ugate. Reduction with sodium metabisulfite or sodium borohydride is not req uired to stabilize the conjugate. Though glycoprotein detection may be perf ormed on transfer membranes, direct detection in gels avoids electroblottin g and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide f ollowed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection w ith horseradish peroxidase or alkaline phosphatase conjugates of streptavid in, which require more than eight hours to complete. Pro-Q Emerald 300 dye- labeled gels and blots may be post-stained with SYPRO Ruby dye, allowing se quential 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 e mits at 610 nm (red), As little as 300 pg of alpha1-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 de tectable 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-dimensi onal gels and on polyvinylidene difluoride membranes.