Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots

Abstract

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.

Characterization of the Human Cervical Mucous Proteome

“…Unambiguous spot matching of phosphoproteins and glycoproteins to the total proteins can be made by direct comparison with the total protein profile in the same gel and low nanogram sensitivity can be achieved. In comparison with other techniques such as radioisotope labeling and immunodetection with antibodies, the fluorescent dye staining method provides a safe, simple, and streamlined way for highly sensitive detection of protein phosphorylation and glycosylation in a complex sample [27]. The existence of phospho- and glycoproteins in the cervical mucous proteome was implicated by prior detection of some glycoproteins, protein kinases and phosphatases in this study (Table 1).…”

Methods for samples preparation in proteomic research

“…Some of the fluorescent dyes have been designed for detection of the post-translational modifications [296,297].…”

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

“…Recently, we described dichromatic methods for detecting glycoproteins and total protein in the same polyacrylamide gel [35]. Other dichromatic methods for detecting All gels were imaged under identical conditions and displayed using identical gray-scale ranges.…”

“…Glycoproteins were detected using Pro-Q Emerald 300 glycoprotein gel stain kit (Molecular Probes) as previously described [35]. Briefly, gels were incubated in 50% methanol for 30-45 min, washed twice in 3% acetic acid for 5-10 min each, and glycoproteins were then oxidized by incubation in 1% periodic acid, 3% acetic acid for 20-30 min.…”

“…Exposure times were generally 1 to 8 s. RuBPSA was imaged in an identical manner as SYPRO Ruby protein gel stain on the indicated instruments. Fluorescence detection of glycoproteins and b-glucuronidase activity was performed as described previously [26,35,36]. Briefly, either 490 nm long pass or 520 nm band pass emission filters are suitable for imaging these dyes.…”