Glycobiology Advance Access originally published online on November 1, 2002
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Glycobiology, 2003, Vol. 13, No. 1 1G-3G
© 2003 Oxford University Press
GLYCO-FORUM SECTION |
Alternative sources of reagents and supplies for fluorophore-assisted carbohydrate electrophoresis (FACE)
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, USA
Received on September 3, 2002; revised on September 6, 2002; accepted on September 6, 2002
Introduction
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a simple, sensitive, and versatile method for analysis of both monosaccharides and oligosaccharides and has become an important method in glycobiology. Recently, the major provider of reagents and supplies for FACE has ceased production and shipping. To assist those who have relied on this provider for FACE reagents and supplies, the editors have asked us to summarize alternative commercial sources and procedures that we have used (Gao and Lehrman, 2002a
,b). Our procedures are modified from those published previously (Jackson, 1994; Starr et al., 1996).
Oligosaccharide profiling gels
Reagents
ANTS (8-aminonophthalene-1,3,6-trisulfonate, catalog A-350) and AMAC (2-aminoacridone, A-6289) are from Molecular Probes (Eugene, OR). ANDS (7-amino-1,3-naphthalenedisulfonic acid, 14644-7) and sodium cyanoborohydride (15615-9) are from Aldrich Chemical (Milwaukee, WI).
Fluorescent oligosaccharide standards
A mild acid hydrolysate (0.1 M HCl, 50°C, 1 hr) of glycogen results in a useful ladder of 
1,4-linked glucose oligomers. Starch can be substituted if one is concerned about interference from 1,6-branched structures (Jackson, 1994). After labeling with ANTS, these mixtures resulted in patterns highly similar to prelabeled standards offered by the major supplier.
Gel electrophoresis apparatus
Gels are sandwiched between 1.0-mm-thick glass plates approximately 10 cm wide, 10 cm long and formed with thin (0.5 mm) spacers to facilitate cooling. Combs with eight 8-mm-wide teeth (2 mm between teeth) are typically used to form loading wells, but 12-tooth combs can also be used. These items can be prepared by a competent machine shop, or similar sized plates, spacers, and combs can be purchased from gel electrophoresis manufacturers. Gels should be cooled to approximately 4°C during electrophoresis; a suitable electrophoresis unit designed for recirculation of coolant appears to be available from Owl Separation Systems (Portsmouth, NH; model P8DS with optional 0.5-mm spacers and custom-ordered 1.0-mm-thick plates).
Imager
Many of the available fluorescence imagers using CCD cameras are suitable for image acquisition and analysis. We have used the Bio-Rad Fluor-S Multi-imager (Bio-Rad Laboratories, Hercules, CA) with a 530DF60 filter and Quantity-One software supplied with the imager.
Derivatization of oligosaccharides with ANTS andANDS
- Prepare a 0.15 M solution of ANTS or ANDS in 15% (v/v) acetic acid. To facilitate dissolution, first prepare a 0.05 M solution of the granular material in 15% acetic acid and lyophilize to dryness. The finely textured dry compound will readily dissolve in 15% acetic acid at a concentration of 0.15 M. These solutions can be stored at -80°C at least 2 months. ANDS is more suitable for our imager filter and in our hands is fivefold more sensitive than ANTS. Prepare a 1 M solution of sodium cyanoborohydride in dimethyl sulfoxide (DMSO), which can be stored at -80°C for at least 2 months.
- Dry the oligosaccharide samples in a centrifugal vacuum device in a 1.5-ml microcentrifuge tube (use a 0.5-ml tube if the sample is below 200 pmol).
- Add 5 µl (or 1 µl if the sample is below 200 pmol) of ANTS or ANDS solution. Add 5 µl (or 1 µl if the sample is below 200 pmol) of sodium cyanoborohydride solution. Mix gently. Centrifuge briefly, and react at 37°C for 18 h.
- Repeat Step 2 above. Dissolve in the desired volume of water.
Preparation of polyacrylamide gels for oligosaccharide profiling
The following solutions are prepared with electrophoresis-quality reagents:
- Resolving gel stock solution: 38% (w/v) acrylamide and 2% (w/v) N,N'-methyllenebisacrylamide
- Stock resolving gel buffer, 8x: 1.5 M Tris-Cl, pH 8.9
- Stacking gel stock solution: 10% (w/v) acrylamide and 2.5% (w/v) N,N'-methyllenebisacrylamide
- Stock stacking gel buffer, 8x: 1.0 M Tris-Cl, pH 6.8
To eliminate particles that emit interfering fluorescence signals during imaging, the above solutions are passed through 0.45-µm filters.
- Stock electrode buffer, 10x: 1.92 M glycine, 0.25 M Tris base, pH 8.3
- 10% ammonium persulfate (prepared daily)
- 100% N,N,N'N'-tetramethylenediamine (TEMED)
- Assemble the gel-casting apparatus and prepare the resolving gel solution by mixing resolving gel stock solution (4 ml); stock resolving gel buffer, 8x (1 ml); water (3 ml); and 10% ammonium persulfate (30 µl).
- Polymerization is initiated by addition of TEMED (10 µl), and the solution is poured into the casting apparatus to a height of 0.5 cm below the bottom of the teeth of the comb. Immediately overlay the gel solution with 1 cm of water. Polymerization occurs after 15 min.
- Prepare the stacking gel solution by mixing stacking gel stock solution (2 ml); stock stacking gel buffer, 8x(0.5 ml); water (1.5 ml); and 10% ammonium persulfate (20 µl). Polymerization is initiated by adding TEMED (5 µl).
- Pour off the water overlay, and fill the remaining space in the mold with stacking gel solution. Insert a comb to form sample wells. The stacker should polymerize within 15 min.
Electrophoretic separation of ANTS- and ANDS-labeled oligosaccharides
- Dilute the stock electrode buffer 10-fold and cool to 4°C. Pour into the electrophoresis apparatus (anode compartment), connected to a circulating cooler set to the appropriate temperature to maintain the gel at 4°C.
- Insert the gel sandwich into the apparatus. Add electrode buffer to the cathode compartment. Mix the sample (dissolved in water) with an equal volume of 2x loading buffer (0.01% thorin I (Aldrich 10456-6) in 20% glycerol). Apply 2–4 µl of mixture to the wells with flat-end tips.
- Connect to a power supply and set it to a constant current of 15 mA (a voltage in the range of 200–1200 V will result). Run until the thorin I marker dye exits the bottom of the gel, usually in 1 h. Turn off the current, remove the gel, and place in the gel imager with both glass plates still attached. A delay of more than 20 min may result in some diffusion of the fluorescent bands.
Monosaccharide composition gels
The oligosaccharide profiling procedure is modified as follows to determine monosaccharide compositions:
Derivatization of monosaccharides with AMAC
Replace the ANTS/ANDS solutions with a 0.1 M solution of AMAC in DMSO containing 15% (v/v) acetic acid. This can be stored at -80°C at least 2 months. Dry the monosaccharide samples and derivatize as described for oligosaccharide profiling. Dissolve the derivatized sample in the desired volume of 30% (v/v) DMSO in water.
Preparation of polyacrylamide gels for monosaccharide composition
Use these solutions in place of those specified for oligosaccharide profiling:
- Stock resolving gel buffer, 4x: 0.75 M Tris–0.5 M boric acid, adjusted to pH 7.0 with concentrated HCl
- Stock stacking gel buffer, 4x: 0.5 M Tris–0.5 M boric acid, adjusted to pH 6.8 with concentrated HCl
- Stock electrode buffer, 5x: 0.5 M glycine, 0.6 M Tris base, and 0.5 M boric acid, final pH 8.3
- Assemble the gel-casting apparatus and prepare resolving gel solution by mixing resolving gel stock solution (4 ml); stock resolving gel buffer, 4x (2 ml); water (2 ml); and 10% ammonium persulfate (30 µl).
- Polymerization and formation of the resolving gel is done as for oligosaccharide profiling.
- Prepare the stacking gel solution by mixing stacking gel stock solution (2 ml); stacking resolving gel buffer, 4x (1 ml); water (1 ml); and 10% ammonium persulfate (15 µl).
- Polymerization and formation of the stacking gel is performed as for oligosaccharide profiling.
Electrophoretic separation of AMAC-labeled monosaccharides
- Dilute the stock electrode buffer fivefold and cool to 4°C. Assemble and cool the electrophoresis apparatus as for oligosaccharide profiling.
- Mix the sample (dissolved in 30% DMSO) with an equal volume of 2x loading buffer. Apply 1–2 µl to the wells.
- Set a constant current to 20 mA (a voltage in the range of 200–1200 V will result). The thorin I marker dye exits the bottom of the gel after about 1 h. Continue for an additional 1–3 h, checking the gel periodically with a long-wave hand-held UV lamp until the fastest-migrating band is near the bottom of the gel. Turn off the current and acquire the gel image as for oligosaccharide profiling.
Examples of oligosaccharide profiling gels prepared by these procedures have been published (Gao and Lehrman, 2002a,b). Examples of monosaccharide composition gels are shown in Figure 1.
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Acknowledgements
Work in our laboratory was supported by NIH grant GM38545 and Welch grant I-1168. We thank Sam Turco, Deborah Sullivan, and Ron Schnaar for valuable comments on the text and experimental evaluation of the methods.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: mark.lehrman{at}utsouthwestern.edu 
Abbreviations
AMAC, 2-aminoacridone; ANDS, 7-amino-1,3-naphthalenedisulfonic acid; ANTS, 8-aminonophthalene-1,3, 6-trisulfonate; DMSO, dimethyl sulfoxide; FACE, fluorophore-assisted carbohydrate electrophoresis; TEMED, N,N,N'N'-tetramethylenediamine.
References
Gao, N. and Lehrman, M.A. (2002a) Analyses of dolichol pyrophosphate-linked oligosaccharides in cells and tissues by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology, 12, 353–360.
Gao, N. and Lehrman, M.A. (2002b) Coupling of the dolichol-P-P-oligosaccharide pathway to translation by perturbation-sensitive regulation of the initiating enzyme, GlcNAc-1-P transferase. J. Biol. Chem., 277, 39425–39435.
Jackson, P. (1994) High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol., 230, 250–265.[Web of Science][Medline]
Starr, C.M., Masada, R.I., Hague, C., Skop, E., and Klock, J.C. (1996) Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates. J. Chromatogr. A, 720, 295–321.[CrossRef][Web of Science][Medline]
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