Cellulose Acetate Electrophoresis of Glycosaminoglycans

Yanusz Wegrowski and Francois-Xavier Maquart

1. Introduction

Electrophoresis on cellulose acetate membrane (zone electrophoresis) is a common method for qualitative and semiquantitative analysis of glycosaminoglycan (GAG) mixtures. The advantage of this method is its simplicity, rapidity, the possibility of processing several samples at the same time, and the low cost of analysis. Apart from cellulose acetate strips and electrophoresis apparatus, usually applied in diagnostic laboratories for serum protein electrophoresis, no special equipment is needed (see Note 1). Several original papers and book chapters describe this technique in different running conditions; the most common is a monodimensional electrophoresis in bivalent cation buffer or pyridine/formiate buffer (1-3). However, no simple system can separate all known GAGs in one run. The separation of different GAGs in one dimension can be done by a several steps method described by Hopwood and Harrison (4), requiring careful temperature control and selective ethanol precipitation after each run. Alternatively, the GAGs can be separated by a two-dimensional method (5), but only one sample at once may be applied on the cellulose acetate sheet. The scope of this chapter is to describe a simple method for rapid separation and visualization of the most common GAGs from tissue or cell culture. Comparison of electrophoretic profiles before and after selective enzymatic treatment (see Chapters 32-36) or HNO2 depolymerization of heparin/heparan sulfate (6) allows characterization in a single run of the major fractions of GAGs in a given sample.

Glycosaminoglycans should be displaced from the protein core of proteoglycans before analysis. This can be done by exhaustive digestion of proteins with nonspecific proteases such as pronase or papain (7). Apart from corneal keratan sulfate, which is bound to protein via an N-glycosyl linkage between N-acetylglucosamine and the amide group of asparagine, O-glycosyl covalent bonding can be alternatively disrupted by a beta-elimination reaction, which liberates all linked GAGs from the protein core (8). The GAG samples have to be desalted and sufficiently concentrated (see Note 2). Tissue

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

or body fluids usually contain sufficient amounts of GAGs to be detected by cationic dye staining without radioactive labeling (see Chapter 16). The limit of Alcian blue staining in the case of electrophoresis is about 100 ng of individual GAG. The techniques that use Safranin O (9) or ruthenium red (10) staining have a detection limit even 100-fold lover, but they are complicated to use. When working with cell culture, one can easily radiolabel the GAGs with 35S-sulfate and/or 3H-glucosamine. Tritium labeling also permits the study of nonsulfated GAGs, such as hyaluronan or chon-droitin. The methods for 35S-sulfate labeling as well as beta-elimination are described elsewhere (8).

We present here two simple variants of cellulose acetate electrophoresis. The first one, published originally by Wessler (11), permits the separation of hyaluronan, heparan sulfate, galactosaminoglycans (dermatan and chondroitin sulfates), and hep-arin in 0.1 M HCl. The second one was used to separate galactosaminoglycans in 0.1 M zinc acetate, pH 5.1. If the electrophoresis is performed first in zinc acetate and then in HCl, the separation of five glycosaminoglycans can be done. The simple method of selective detection of 35S-sulfate vs 3H-glucosamine is also described.

2. Materials

2.1. Electrophoresis of Glycosaminoglycans

1. Cellulose acetate strips and electrophoresis apparatus (e.g., Titan III cellulose acetate plates and Titan Zip Zone Chamber from Helena Research Laboratories, Beaumont, TX, USA, or Sebiagel® and Electrophoresis Apparatus from Sebia, Issy-les-Moulineaux, France).

2. Glycosaminoglycan standards (Sigma, St. Louis, MO, USA). Dissolve each glycosaminoglycan in deionized water at concentration 1 mg/mL. This is stable indefinitely if frozen.

3. 0.1 M zinc acetate buffer, acidified to pH 5.1 with acetic acid.

5. Absolute ethanol.

6. Phenol red (Sigma). Saturated aqueous solution.

7. A microsyringue for sample application (e.g., Hamilton).

2.2. Staining with Alcian Blue

1. Stock solution of Alcian blue 8GX (Sigma): Prepare 0.4% (m/v) solution in absolute ethanol. Filter through cotton or Kleenex or Büchner funnel. Stable indefinitely in the dark.

2. Staining buffer: 0.05 M Natrium acetate containing 0.1 M MgCl2, pH 5.8. Sodium azide 0.05 % (m/v), may be added to prevent bacterial development. Store at 4°C.

3. Staining solution: Mix 1-to-1 stock Alcian solution with staining buffer. May be reused several fold until the formation of precipitate.

4. Destaining solution: Mix 1-to-1 staining buffer with ethanol.

2.3. Autoradiography of Glycosaminoglycans

1. Glycerol 2% (v/v) in absolute ethanol.

2. PPO solution: Dissolve PPO (2,5-diphenyloxasol, Merck, Darmstadt, Germany), 2% (m/v) in glycerol-containing ethanol.

3. Autoradiography cassette with intensifying screens and autoradiography film.

4. Saran Wrap foil.

5. Developing facilities.

3. Method

3.1. Electrophoresis

1. Soak cellulose acetate membrane for at least 10 min in electrophoresis buffer with gentle agitation (see Note 3).

2. Fill the electrode chambers with the same buffer.

3. Blot the membrane in filter paper to remove the excess of liquid. Do not dry. Immediately install in electrophoresis apparatus (see Note 4).

4. Starting 8 mm from one border and about 1 cm from cathode (-) end, mark with a phenol red a series of 5-mm traits indicating the places for sample loading. Leave a space of 5 mm between the traits. Let the liquid dry.

5. Apply samples or standards by portions not exceeding 3 ^L. Let the liquid dry every time. Optimal loading quantity for staining is the equivalent of 1 ^g of standard GAG and, for autoradiography, 40,000 cpm as 35S-sulfate.

6. Run the electrophoresis at room temperature for 2 h at constant voltage. The current at the beginning should not exceed 1 mA/cm of width for zinc acetate or 1.5 mA/cm for HCl (see Note 5).

7. If the electrophoresis is performed in 0.1 M HCl, fix the membrane (see step 9). If it is done in zinc acetate buffer, remove the membrane and soak it for about 1 min in 0.1 M HCl. At the same time, fill the buffer tanks of apparatus with 0.1 M HCl.

8. Blot and reinstall the membrane. Continue the electrophoresis for 1 h at the same current.

9. After electrophoresis, the GAGs are fixed by soaking the membrane for about 1 min in absolute ethanol.

3.2. Staining

1. Soak the membrane in Alcian blue staining solution for 30 min with gentle agitation.

2. Wash the membranes in three baths of destaining solution (see Note 6).

3. Wash the membranes in water if scanning or photography have to be performed. The color is stable for several days. Figure 1a shows an example of electrophoresis of tissue GAGs performed in zinc acetate/HCl solutions, followed by Alcian blue staining.

4. To dry the membranes, place them in anhydrous methanol for 1 min with gentle agitation, then transfer for exactly 1 min to freshly prepared 18% (v/v) acetic acid in methanol. The membranes are removed, deposited on glass plates, and dried at 80°C for 10 min. After cooling at room temperature, the membranes are unstuck from the glass plate with a spatula or scalpel. They may be stored indefinitely.

3.3. Autoradiography

1. Wash the membrane containing the radiolabeled GAGs in ethanolic glycerol. If the labeling was performed with only one isotope, wash the membrane directly in PPO solution (see step 2). Dry between Whatman filter papers. Install in cassette. Cover with Saran foil and autoradiography film in a dark room. Keep at -80°C for 24 h for 40,000 cpm as 35S-sulfate deposited. Less isotope needs longer exposition time. Develop the film. Figure 1b shows an example of electrophoresis in 0.1 M HCl and direct autoradiography.

2. For tritium autoradiography, wash the membrane in PPO solution. Dry and reexpose as above. (Fig. 1c).

3. Optionally, after autoradiography, the bands can be excised, dissolved in xylene, and counted by liquid scintillation.

Fig. 1. Examples of electrophoresis in zinc acetate and HCl of tissue GAGs (A) and electrophoresis in HCl of 35S-sulfate and 3H-glucosamine labeled GAGs extracted from MRC5 fibroblast's culture medium (B,C). Panel (A) shows the separation of 1 ^g (as uronic acid) of GAGs from fibrous tissue obtained from patient with type IV Ehlers-Danlos syndrome (1,2) and control fibrous tissue (3,4) before (1,3) and after (2,4) chondroitinase AC treatment (12). The GAGs were stained with Alcian blue. Note the higher proportion of dermatan sulfate in control tissue. The panels at right (B,C) show the electrophoresis of the secreted, radiolabeled GAGs from control cells (5,5') and the cells stimulated with 5 ng/mL each of TGFp and EGF (6,6'). Ten thousand cpm of 35S-sulfate were deposited on each lane. The electrophoresis was autoradiographied before (B) and after (C) impregnation with 2% (m/v) PPO. The migration position of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and hyaluronan (HA) standards are indicated on the margins. The bi-directional arrow indicates the application points. Anode and cathode extremities are indicated by (+) and (-), respectively.

Fig. 1. Examples of electrophoresis in zinc acetate and HCl of tissue GAGs (A) and electrophoresis in HCl of 35S-sulfate and 3H-glucosamine labeled GAGs extracted from MRC5 fibroblast's culture medium (B,C). Panel (A) shows the separation of 1 ^g (as uronic acid) of GAGs from fibrous tissue obtained from patient with type IV Ehlers-Danlos syndrome (1,2) and control fibrous tissue (3,4) before (1,3) and after (2,4) chondroitinase AC treatment (12). The GAGs were stained with Alcian blue. Note the higher proportion of dermatan sulfate in control tissue. The panels at right (B,C) show the electrophoresis of the secreted, radiolabeled GAGs from control cells (5,5') and the cells stimulated with 5 ng/mL each of TGFp and EGF (6,6'). Ten thousand cpm of 35S-sulfate were deposited on each lane. The electrophoresis was autoradiographied before (B) and after (C) impregnation with 2% (m/v) PPO. The migration position of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and hyaluronan (HA) standards are indicated on the margins. The bi-directional arrow indicates the application points. Anode and cathode extremities are indicated by (+) and (-), respectively.

4. Notes

1. Any horizontal electrophoresis apparatus may be easily adapted to support cellulose acetate strips. The strips are lying in the gel platform and are connected with the electrode chambers (buffer tanks) by Whatman no. 1 filter paper. Do not submerge the membrane.

2. The salts provoke lateral diffusion of GAGs. It can be easily eliminated by dialysis or gel-filtration. Precipitated GAGs can be desalted by successive washing with 90% ethanol and absolute ethanol, drying, and dissolving in water. For practical reasons, maximal volume for electrophoresis should not excess 10 ^L.

3. Use zinc acetate or HCl as electrophoresis buffer. Keep the liquid at 4°C before electrophoresis.

4. The membranes do not dry in the electrophoresis tank when the extremities sink in the buffer.

5. The electrophoresis in zinc acetate buffer is performed at 80 V for a membrane of 10 cm length and that performed in 0.1 M HCl at 40 V for the same membrane dimension.

6. If necessary, the membrane can be destained overnight at room temperature in a covered tank, to avoid ethanol evaporation.

References

1. Morris, J. E., Canoy, D. W., and Rynd, L. S. (1981) Electrophoresis with two buffers in one dimension in the analysis of glycosaminoglycans on cellulose acetate strips. J. Chromatogr. 224, 407-413.

2. Beeley, J. C. (1985) Characterization of charge, in Glycoprotein and Proteoglycan Techniques (Burdon, R. H. and van Knippenberg, P. H. eds.), Elsevier, Amsterdam, The Netherlands, pp. 88-94.

3. Kodama, C., Kodama, T., and Yosizawa, Z. (1988) Methods for analysis of urinary glycosaminoglycans. J. Chromatogr. 429, 293-313.

4. Hopwood, J. J. and Harrison, J. R. (1982) High-resolution electrophoresis of urinary gly-cosaminoglycans: an improved screening test for the mucopolysaccharidoses. Anal. Biochem. 119, 120-127.

5. Hata, R. and Nagai, Y. (1973) A micro-colorimetric determination of acidic glycosaminoglycans by two dimensional electrophoresis on a cellulose acetate strip. Anal. Biochem. 52, 652-656.

6. Cifonelli, J. A. (1976) Nitrous acid depolymerisation of glycosaminoglycans. Meth. Carbohydr. Chem. 7, 139-142.

7. Breen, M., Weinstein, H. G., Blacik, L. J., Borcherding, M. S., and Sittig, R. A. (1976) Microanalysis and characterization of glycosaminoglycans from human tissue via zone electrophoresis. Meth. Carbohydr. Chem. 7, 101-115.

8. Rider, C. C. (1998) Analysis of glycosaminoglycans and proteoglycans, in Glycoanalysis Protocols (Hounsell, E. F., ed.), Humana, Totowa, NJ, pp. 131-143.

9. Lammi, M. and Tammi, M. (1988) Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O. Anal. Biochem. 168, 352-357.

10. Gaffen, J. D., Price, F. M., Bayliss, M. T., and Mason, R. M. (1994) A ruthenium-103 red dot blot assay specific for nanogram quantities of sulfated glycosaminoglycans. Anal. Biochem. 218, 124-130.

11. Wessler, E. (1971) Electrophoresis of acidic glycosaminoglycans in hydrochloric acid: a micro-method for sulfate determination. Anal. Biochem. 41, 67-69.

12. Wegrowski, Y., Bellon, G., Quereux, C., and Maquart, F. X. (1999) Biochemical alterations of uterine leiomyoma extracellular matrix in type IV Ehlers-Danlos syndrome. Am. J. Obstet. Gynecol. 180, 1032-1034.

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  • salvatore
    What van be used to buffer cellulose acetate?
    8 months ago

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