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
Preparation of polyacrylamide gels for oligosaccharide profiling
The following solutions are prepared with electrophoresis-quality reagents:
To eliminate particles that emit interfering fluorescence signals during imaging, the above solutions are passed through 0.45-µm filters.
Electrophoretic separation of ANTS- and ANDS-labeled oligosaccharides
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:
Electrophoretic separation of AMAC-labeled monosaccharides
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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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, 353360.
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, 3942539435.
Jackson, P. (1994) High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol., 230, 250265.[ISI][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, 295321.[CrossRef][ISI][Medline]