Fluorescence Detection of Glycoproteins in Gels and on Electroblots

Wayne F. Patton1

1 Molecular Probes, Inc., Eugene, Oregon
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.8
DOI:  10.1002/0471143030.cb0608s16
Online Posting Date:  November, 2002
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Abstract

Glycosylation of proteins is a dynamic post‐translational modification commonly found in eukaryotes, changing with development, differentiation, and disease state. Identification of glycoproteins is important for completely characterizing the population of proteins found at a given point in time. This unit contains protocols for identifying glycoproteins in general using a fluorescence based periodate/Schiff base method and those containing specific carbohydrates using a lectin‐based fluorescent method.

     
 
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Table of Contents

  • Basic Protocol 1: Fluorescent Detection of Glycoproteins in Polyacrylamide Gels
  • Alternate Protocol 1: Fluorescent Detection of Glycoproteins on Electroblot Membranes
  • Basic Protocol 2: Fluorescent Detection of Glycoproteins Containing Terminal α‐Mannopyranosyl and α‐Glucopyranosyl Residues on Electroblot Membranes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescent Detection of Glycoproteins in Polyacrylamide Gels

  Materials
  • Protein sample of interest
  • Fix solution (see recipe)
  • Wash solution (see recipe)
  • Pro‐Q Emerald 300 Glycoprotein Gel Stain Kit (Molecular Probes) containing:
  •  50× Pro‐Q Emerald 300 reagent, concentrate in DMF
  •  Pro‐Q Emerald 300 dilution buffer
  •  Periodic acid (oxidizing reagent; see recipe)
  •  CandyCane glycoprotein molecular weight standards (see recipe), sufficient volume for approximately 20 gel lanes
  •  SYPRO Ruby protein gel stain
  • Deionized, high quality water
  • 10% (v/v) methanol or ethanol, spectroscopy grade (optional)
  • 7% (v/v) glacial acetic acid (optional)
  • Polystyrene staining dishes (e.g., a weighing boat for minigels or larger container for larger gels)
  • Orbital shaker
  • UV transilluminator
  • Photographic camera or CCD camera and appropriate filters
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (unit 6.1)

Alternate Protocol 1: Fluorescent Detection of Glycoproteins on Electroblot Membranes

  Materials
  • Protein sample of interest
  • PVDF membrane
  • Fix solution (see recipe)
  • Wash solution (see recipe)
  • Pro‐Q Emerald 300 Glycoprotein Blot Stain Kit (Molecular Probes) containing:
  •  50× Pro‐Q Emerald 300 reagent, concentrate in DMF
  •  Pro‐Q Emerald 300 dilution buffer
  •  Periodic acid (oxidizing solution; see recipe)
  •  CandyCane glycoprotein molecular weight standards (see recipe), sufficient volume for ∼20 gel lanes
  •  SYPRO Ruby protein blot stain
  • Methanol, spectroscopy grade (optional)
  • Glacial acetic acid (optional)
  • 95°C heat block
  • Polystyrene staining dishes (e.g., weighing boat for minigels or larger containers for larger gels)
  • Orbital shaker
  • UV epi‐illuminator
  • Photographic camera or CCD camera and appropriate filters
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (unit 6.1) and electroblotting (unit 6.2)

Basic Protocol 2: Fluorescent Detection of Glycoproteins Containing Terminal α‐Mannopyranosyl and α‐Glucopyranosyl Residues on Electroblot Membranes

  Materials
  • Protein samples of interest
  • PVDF membrane
  • 50% methanol
  • Wash solution II (see recipe)
  • Blocking solution (see recipe)
  • Pro‐Q Glycoprotein Blot Stain Kit with Concanavalin A (Molecular Probes) containing:
  •  Concanavalin A, alkaline phosphatase conjugate (Con A‐AP) stock solution  (see recipe)
  •  DDAO phosphate stock solution (see recipe)
  •  Dimethylformamide (DMF)
  •  CandyCane glycoprotein molecular weight standards (see recipe), sufficient  volume for ∼20 gel lanes
  • Incubation buffer (see recipe)
  • 10 mM Tris/1 mM MgCl 2, pH 9.5
  • Polystyrene staining dishes (e.g., weigh boat for minigel or larger container for larger gels)
  • Plastic wrap
  • UV epi‐illumination and a digital or film camera, or a laser equipped with a 633‐nm helium‐neon laser or 635‐nm diode laser source
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (unit 6.1), electroblotting procedures (unit 6.2), and SYPRO Ruby protein blot staining (see protocol 2)
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Figures

Videos

Literature Cited

Literature Cited
   Beeley, J. 1985. Glycoproteins and proteoglycan techniques. In Laboratory Techniques in Biochemistry and Molecular Biology, (R. Burdon and P. van Knippenberg eds.) vol. 16, pp. 5‐28. Elsevier Press, New York.
   Hirabayashi, J., Arata, Y., and Kasai, K. 2001. Glycome project: Concept, strategy and preliminary application to Caenorhabditis elegans. Proteomics 1:285‐294.
   Koch, G. and Smith, M. 1990. The analysis of glycoproteins in cells and tissues by two‐dimensional polyacrylamide gel electrophoresis. Electrophoresis 11:213‐219.
   Koketsu, M. and Linhardt, R. 2000. Electrophoresis for the analysis of acidic oligosaccharides. Anal. Biochem 283:136‐145.
   Packer, N., Pawlak, A., Kett, W., Gooley, A., Redmond, J., and Williams, K. 1997. Proteome analysis of glycoforms: A review of strategies for the microcharacterization of glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis. Electrophoresis 18:452‐460.
   Packer, N., Ball, M., and Devine, P. 1999. Glycobiology and proteomics. In 2‐D Proteome Analysis Protocols, Methods in Molecular Biology, (A. Link, ed.) vol. 112, pp.341‐352. Humana Press, Totowa, N.J.
   Patton, W. 2000a. A thousand Points of light; The application of fluorescence detection technologies to two‐dimensional gel electrophoresis and proteomics. Electrophoresis 21:1123‐1144.
   Patton, W. 2000b. Making blind robots see; The synergy between fluorescent dyes and imaging devices in automated proteomics. BioTechniques 28:944‐957.
   Raju, T. 2000. Electrophoretic methods for the analysis of N‐linked oligosaccharides. Anal. Biochem. 283:125‐132.
   Reuter, G. and Gabius, H. 1999. Eukaryotic glycosylation: Whim of nature or multipurpose tool?. Cell Mol. Life Sci. 55:368‐422.
   Steinberg, T., Pretty On Top, K., Berggren, K., Kemper, C., Jones, L., Diwu, Z., Haugland, R., and Patton, W. 2001. Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots. Proteomics 1:841‐855.
   Taverna, M., Tran, N., Merry, T., Horvath, E., and Ferrier, D., 1998. Electrophoretic methods for process monitoring and the quality assessment of recombinant glycoproteins. Electrophoresis 19:2572‐2594.
Key References
   Steinberg et al. 2001. See above
  Describes detection of glycoproteins in gels and on blots using Pro‐Q Emerald 300 Glycoprotein Detection Kits as well as the detection of concanavalin A–binding and wheat germ agglutinin–binding glycoproteins on blots using lectin–alkaline phosphatase conjugates and DDAO phosphate.
   Berggren, K., Steinberg, T., Lauber, W., Carroll, J., Lopez, M., Chernokalskaya, E., Zieske, L., Diwu, Z., Haugland, R., and Patton, W. 1999. A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports. Anal. Biochem. 276:129‐143.
  Describes counter‐staining with SYPRO Ruby dye for the detection of total protein profiles on electroblot membranes.
   Berggren, K., Chernokalskaya, E., Steinberg, T., Kemper, C., Lopez, M., Diwu, Z., Haugland, R., and Patton, W. 2000. Background‐free, high‐sensitivity staining of proteins in one‐ and two‐dimensional sodium dodecyl sulfate–polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis 21:2509‐2521.
  Describes counter‐staining with SYPRO Ruby dye for the detection of total protein profiles in polyacrylamide gels.
   Lopez, M., Berggren, K., Chernokalskaya, E., Lazarev, A., Robinson, M., and Patton, W. 2000. A comparison of silver stain and SYPRO Ruby protein gel stain with respect to protein detection in two‐dimensional gels and identification by peptide mass profiling. Electrophoresis 21:3673‐3683.
  Describes optimized methods for protein identification by matrix‐assisted laser desorption time‐of‐flight mass spectrometry after staining gels with SYPRO Ruby dye.
Internet Resources
  http://www.cbs.dtu.dk/databases/OGLYCBASE/
  O‐GLYCBASE; a database of 198 glycoprotein entries with experimentally verified O‐glycosylation site information.
  http://www.glycosuite.com
  GlycoSuite; a relational database that curates information from the scientific literature on glycoprotein derived glycan structures, their biological sources, the references in which the glycan was described, and the methods used to determine the glycan structure.
  http://www.expasy.ch/tools/glycomod/
  GlycoMod; a software tool designed to find all possible compositions of a glycan structure from its experimentally determined mass.
  http://www.probes.com
  Molecular Probes commercial Web site containing information about fluorescence detection technologies, including glycoprotein, total protein, lipopolysaccharides, and nucleic acids.
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