Chapter 16 Glycosaminoglycan Blotting and Detection After Electrophoresis Separation Nicola Volpi and Francesca Maccari Abstract Separation of glycosaminoglycans (GAGs) by electrophoresis and their characterization to the microgram level are integral parts of biochemical research. Their blotting on membranes after electrophoresis offers the advantage to perform further analysis on single separated species such as identification with antibodies and/or recovery of single band. A method for the blotting and immobilizing of several nonsulfated and sulfated complex GAGs on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose-gel electrophoresis is illustrated. This approach to the study of these complex macromolecules utilizes the capacity of agarose-gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses. Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of GAGs are capillary blotted after their separation in agarose-gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100 % and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 μg. Nonsulfated polyanions, for example hyaluronic acid (HA), may also be transferred to membranes with a limit of detection of approximately 0.1–0.5 μg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes used for immunological detection or other applications. Key words Glycosaminoglycans, Electrophoresis, Blotting, Heparin, Chondroitin sulfate
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Introduction Blotting of (macro) molecules on membranes after their separation by electrophoresis takes advantage of the possibility to perform further analysis on single separated species by several approaches, e.g., specific binding and identification with antibodies or recovery of single band, and can be used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are linear, unbranched, complex heteropolysaccharides composed of a variable number of repeating disaccharide units. Each disaccharide consists of one hexosamine,
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_16, © Springer Science+Business Media New York 2015
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Nicola Volpi and Francesca Maccari D-galactosamine or D-glucosamine and one uronic acid, Dglucuronic acid or L-iduronic acid (IdoA) or neutral hexose, D-galactose in keratan sulfate. According to the type of the monosaccharide units and the glycosidic bonds between them, GAGs can be divided into four main categories: (1) hyaluronic acid or hyaluronan (HA), (2) chondroitin sulfate (CS) and dermatan sulfate (DS), (3) heparan sulfate (HS) and heparin (Hep), and (4) keratan sulfate (KS) (Fig. 1). Separation of GAGs by electrophoresis is routine in many laboratories and their characterization to the microgram level forms an integral part of biochemical research, especially with respect to obtaining information from unknown purified polysaccharides.
Fig. 1 Structures of disaccharides forming GAGs. Major modifications for each structure are illustrated (R = H or SO3−) but minor variations are possible
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This is more important for natural (macro) molecules produced by extraction and purification from different animal tissues having several fundamental biological activities, as well as pharmacological properties, making them important drugs for use in clinical and pharmaceutical fields [1–5]. Cellulose acetate [6], nitrocellulose membrane (NC) [7], agarose-gel [8], and polyacrylamide-gel [9, 10] electrophoretic techniques are generally utilized for GAGs isolation and separation and for qualitative and quantitative analyses of their mixtures or single species. However, agarose-gel electrophoresis permits the separation and identification of several GAG species, such as slow (SM heparin) and fast moving heparin (FM heparin) or heparan sulfate, dermatan sulfate, and chondroitin sulfate [8, 11]. GAGs are strongly hydrophilic and negatively charged macromolecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. As a consequence, membranes have been derivatized with cationic detergents to make them hydrophilic and positively charged, like cetylpyridinium chloride (CPC)-treated NC membranes used in this protocol. After their electrophoretic separation, several intact GAGs with high molecular mass, such as HA, CS, highly sulfated CS, DS, HS, Hep, and its two components, FMHep and SMHep species, were transferred on NC membranes treated with a cationic detergent, CPC [12]. Quantitative analysis was performed after visualization of bands by cationic dyes, the recovery of single molecules released from membrane was also examined [12], and the direct and specific recognition of these polysaccharides by antibodies on CPC-treated NC supports has also been described [13].
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Materials 1. High purity agarose (see Note 1). 2. Barium acetate and NC membranes 0.45 mm, binding capacity of 80–100 μg/cm2. 3. 1,2-Diaminopropane (PDA) and cresol red. 4. Cetylpyridinium chloride (CPC). 5. Toluidine blue, alcian blue, and Whatman 3 MM paper. 6. All the other reagents should be of analytical grade.
2.1 Glycosaminoglycans
1. Different GAGs to be used as standard may be purchased from Sigma-Aldrich (http://www.sigmaaldrich.com) or other specialized companies. 2. Extraction and purification protocols for various GAGs are available in specific scientific articles and monographs [3, 5, 14–21].
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1. 40 mM barium acetate buffer, pH 5.8 with 1 M acetic acid. Store at 4 °C. 2. 50 mM 1,2-diaminopropane (PDA) buffer: Buffered at pH 9 with glacial acetic acid. Store at 4 °C. 3. Cresol red solution: Dissolve 10 mg of the dye in 100 mL of distilled water (final concentration of 0.1 mg/mL). Store at 4 °C.
2.3 Blotting on Membranes
1. 1 % CPC in 30 % 2-propanol. This solution should be always freshly prepared. 2. 150 mM NaCl. 3. Transfer buffer: 100 mM Tris-acetate buffer pH 7.3. Store at 4 °C.
2.4 Membrane Staining
1. Alcian blue solution: Dissolve 50 mg of the dye in 1 mL 8 M guanidine and 19 mL of 18 mM sulfuric acid-0.25 % Triton X-100. This staining reagent is always freshly prepared. 2. Reversible staining is always freshly prepared with toluidine blue. 20 mg toluidine blue is dissolved in 100 mL of 3 % acetic acid.
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Methods
3.1 Agarose-Gel Electrophoresis of Glycosaminoglycans
1. Prepare 0.5 % agarose solution in 40 mM barium acetate buffer (see Note 2). Heat the solution in a microwave oven or on a stirrer mixer, mixing continuously until the agarose completely dissolves and a clear solution is obtained. Do not allow the solution to boil. 2. Thoroughly clean a single glass plate of 7 × 8 cm (of approx. 2 mm thickness) with alcohol. After drying with paper, place it in a holder of approx. 8 × 10 cm and pour the warm agarose solution (see Notes 3 and 4). 3. Prior to electrophoresis, leave the gel at room temperature (RT) for approx. 30 min. Cut the gel by the side of the glass plate and put it with the same glass plate on a grid made of 1 × 1 cm squares. Make four small wells by using a flat chisel of approx. 5 mm, taking care to leave approx. 5 mm between each well (see Note 5). Make the wells approx. 2 cm from the edge of the gel. 4. 10 μL of GAGs standard or samples (see Note 6) may be layered by micropipets into the wells. 5. The electrophoretic run is performed in 50 mM PDA for 150 min at 50 mA by using a Pharmacia Multiphor II electrophoretic cell instrument (see Note 7).
Glycosaminoglycan Blotting
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1. Cut NC membranes in portions of 7 × 8 cm. 2. Wet the membrane in freshly prepared 1 % CPC in 30 % 2-propanol for 5 min. 6 mL of CPC solution should be used. Shake the membrane manually. 3. After wetting, add 50 mL of 150 mM NaCl and incubate the membrane on a shaker for 15 min. 4. CPC-derivatized membrane is rinsed several times in 150 mM NaCl (see Note 8) and then equilibrated with continuous shaking in the same NaCl solution until blotting is performed. 5. After agarose-gel electrophoresis, carefully remove the gel from the glass plate. 6. Prepare the blotting sandwich by assembling a Whatman 3 MM paper immersed in the buffer reservoir (see Note 9). Carefully place the agarose gel on the 3 MM paper with the wells located parallel to the two buffer reservoirs. The detergent-treated NC membrane is then laid on top of the gel (see Note 10). Three further wetted filter papers, two wetted sponges, and 5 cm of absorbent paper tissue are carefully laid on top of the NC membrane, ensuring that no bubbles are trapped in the resulting sandwich. The blotting sandwich is stabilized by putting a 500 g weight on the top. 7. The capillary blotting is performed overnight at RT.
3.3 Staining Procedures
1. Irreversible staining is performed by means of alcian blue. After 2 h staining, membrane is destained by rinsing in 150 mM NaCl until background staining disappears (see Note 11). An example of the results produced is shown in Fig. 2a.
Fig. 2 (a) Decreasing amounts (a: 7.5 μg, b: 5.0 μg, c: 2.5 μg, d: 0.1 μg) of mixtures of GAGs composed of (1) slow moving heparin, (2) fast moving heparin, (3) dermatan sulfate, and (4) chondroitin sulfate electrophoretically separated by agarose-gel and blotted on CPC-treated NC membrane. The membrane was stained with alcian blue. (b) Quantitation of immobilized sulfated and nonsulfated GAGs on CPC-treated NC membranes after agarose-gel electrophoresis, capillary blotting, staining with alcian blue, destaining, and densitometric analysis. Hep heparin, SM Hep slow moving heparin, FM Hep fast moving heparin, DS dermatan sulfate, CSi highly sulfated chondroitin sulfate, HS heparan sulfate, CSb chondroitin sulfate, HA hyaluronic acid, K4 bacterial polysaccharide K4, K4 Defructosylated defructosylated bacterial polysaccharide K4. Reprinted with permission
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2. Irreversible staining with alcian blue can be used for quantitative studies with a high detection sensitivity (see below). Quantitative analysis of GAGs may be performed with a densitometer connected to a computer by using an image processing and analysis software. The wet membranes should be scanned in the RGB mode and saved in gray scale. An example of the results produced is shown in Fig. 2b. 3. GAGs separated by agarose-gel electrophoresis and transferred to a membrane can also be detected by toluidine blue staining (see Note 12). Reversible staining may be obtained by treating membranes with toluidine blue solution for 5 min. The membrane is rinsed for 30–60 s in 3 % acetic acid in the presence of 0.1 % CPC to remove the excess stain, and further utilized (see Notes 13 and 14).
4
Notes 1. Agarose should be of very high quality, suitable to run high resolution gels (possibly certified for molecular biology, ideal for the separation of small DNA fragments). Agarose from Sigma-Aldrich or from Bio-Rad is suitable. 2. The volume of the agarose solution is strictly related to the dimension of the gel. For a gel of 7 × 8 cm with a thickness of about 4–5 mm, a volume of 50 mL (250 mg agarose) is advisable. 3. Carefully eliminate possible air bubbles in the warm solution, and allow the agarose solution to convert into a gel at RT for about 30–60 min. The gel may be stored at 4 °C for approx. 4–5 days after covering it with a plastic sheet. 4. It is very important to consider that a gel having a thickness lower than about 4–5 mm does not permit the layering of the samples. On the contrary, a gel with a greater thickness requires greater migration times. 5. Carefully dry the wells by using little pieces of Whatman 3 MM paper of approx. 5 × 20 mm. 6. GAGs standard should be prepared at a concentration of 0.5 mg/mL in distilled water with a final absolute amount of 5 μg loaded on the gel. Extracted GAGs from different matrices should be quantitatively evaluated by means of known assays [1, 13–15, 17] before performing the electrophoretic separation. The optimum concentration range of unknown purified GAGs loaded on the gel should be from 2 to 8 μg. 7. Due to the possible variability of the electrophoretic conditions, 2 μL of cresol red solution (0.1 mg/mL) should be added to each standard or sample solution (10 μL) in order to
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make a more accurate evaluation of the electrophoretic migration. A good migration time and electrophoretic separation are obtained at a cresol red migration of approx. 20–25 mm. 8. Derivatized membranes should be rinsed several times in 150 mM NaCl with vigorous shaking until no foaming is observed. This step permits the complete removal of excess CPC. 9. The Whatman 3 MM is immersed in the two buffer reservoirs permitting the migration of the buffer from the tank to the top side of the blotting sandwich. As a consequence, make sure that sufficient buffer volume for a complete GAGs migration (approx. 1 L) is available. 10. Carefully remove possible air bubbles entrapped between the gel and the membrane by using a little glass pestle. 11. It is very important to optimize the irreversible staining period to obtain a good band staining against a clear background. Under the experimental conditions described, an optimum staining time would be 2 h. Furthermore, use several changes of the destaining solution to produce the best results. 12. The sensitivity of staining with toluidine blue is about 10–15 times lower than that with alcian blue (an example is illustrated in Fig. 3), but if the membrane is then destained, the same lanes can be used for immunological detection or other applications.
Fig. 3 Quantitation of immobilized sulfated and nonsulfated glycosaminoglycans on CPC-treated NC membranes after agarose-gel electrophoresis, capillary blotting, staining with toluidine blue, destaining, and densitometric analysis. Hep heparin, SM Hep slow moving heparin, FM Hep fast moving heparin, DS dermatan sulfate, CSi highly sulfated chondroitin sulfate, HS heparan sulfate, CSb chondroitin sulfate, HA hyaluronic acid, K4 bacterial polysaccharide K4, K4 Defructosylated defructosylated bacterial polysaccharide K4. Reprinted with permission
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13. After toluidine blue detection, membrane is destained using 3 % acetic acid in the presence of 0.1 % CPC. Under these conditions, the destaining of the bands is completed within 5 min and without any loss of immobilized molecules. 14. The electrophoretically separated GAGs transferred on NC may also be released and recovered from the cationized membranes at the μg level for further analysis, such as disaccharide pattern evaluation, molecular mass determination, and characterization of specifically sulfated sequences inside the polysaccharide chains. The immobilized GAGs are efficiently released from the membrane using a nonionic detergent at high ionic strength (for details see ref. 13). References 1. Mammen EF, Walenga JM, Fareed J (eds) (1991) Development of non-heparin glycosaminoglycans as therapeutic agents, vol 17, Seminars in thrombosis and hemostasis. Thieme Medical Publishers, New York/Stuttgart 2. Ofosu FA, Danishefsky I, Hirsh J (eds) (1989) Heparin and related polysaccharides. Structure and activities, vol 556. Annals of the New York Academy of Sciences, New York 3. Heinegard D, Sommarin Y (1987) Isolation and characterization of proteoglycans. Meth Enzymol 144:319–373 4. Crescenzi V, Dea ICM, Paoletti S, Stivala SS, Sutherland IW (eds) (1989) Biomedical and biotechnological advances in industrial polysaccharides. Gordon and Breach Sc. Pub., New York 5. Volpi N (ed) (2006) Chondroitin sulfate: structure, role and pharmacological activity. Academic, New York 6. Cappelletti R, Del Rosso M, Chiarugi VP (1979) A new electrophoretic method for the complete separation of all known animal glycosaminoglycans in a monodimensional run. Anal Biochem 99:311–315 7. Volpi N (1996) Electrophoresis separation of glycosaminoglycans on nitrocellulose membranes. Anal Biochem 240:114–118 8. Nader HB, Takahashi HK, Guimaraes JA, Dietrich CP, Bianchini P, Osima B (1981) Heterogeneity of heparin: characterization of one hundred components with different anticoagulant activities by a combination of electrophoretic and affinity chromatography methods. Int J Biol Macromol 3:356–360 9. Rice KG, Rottink MK, Linhardt RJ (1987) Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis. Biochem J 244:515–522
10. Lyon M, Gallagher JT (1990) A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver. Anal Biochem 185:63–70 11. Volpi N (1993) “Fast moving” and “slow moving” heparins, dermatan sulfate, and chondroitin sulfate: qualitative and quantitative analysis by agarose-gel electrophoresis. Carbohydr Res 247:263–278 12. Maccari F, Volpi N (2002) Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation. Electrophoresis 23:3270–3277 13. Maccari F, Volpi N (2003) Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose-gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes. Electrophoresis 24:1347–1352 14. Volpi N (ed) (2002) Analytical techniques to evaluate the structure and function of natural polysaccharides, glycosaminoglycans. Research Signpost, India 15. Savolainen H (1999) Isolation and separation of proteoglycans. J Chromatogr B Biomed Sci Appl 722:255–262 16. Beaty NB, Mello RJ (1987) Extracellular mammalian polysaccharides: glycosaminoglycans and proteoglycans. J Chromatogr 418:187–222 17. Roden L, Baker JR, Cifonelli JA, Mathews MB (1972) Isolation and characterization of connective tissue polysaccharides. Meth Enzymol XXVIII:73–140 18. Takegawa Y, Araki K, Fujitani N, Furukawa J, Sugiyama H, Sakai H, Shinohara Y (2011)
Glycosaminoglycan Blotting Simultaneous analysis of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed by single-step zwitterionic-hydrophilic interaction chromatography. Anal Chem 83:9443–9449 19. Zhang F, Sun P, Muñoz E, Chi L, Sakai S, Toida T, Zhang H, Mousa S, Linhardt RJ (2006) Microscale isolation and analysis of
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heparin from plasma using an anion exchange column. Anal Biochem 353:284–286 20. Shao C, Shi X, White M, Huang Y, Hartshorn K, Zaia J (2013) Comparative glycomics of leukocyte glycosaminoglycans. FEBS J 280:2447–2461 21. Mizumoto S, Sugahara K (2012) Glycosaminoglycan chain analysis and characterization (glycosylation/epimerization). Methods Mol Biol 836:99–115
1
Introduction Blotting of (macro) molecules on membranes after their separation by electrophoresis takes advantage of the possibility to perform further analysis on single separated species by several approaches, e.g., specific binding and identification with antibodies or recovery of single band, and can be used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are linear, unbranched, complex heteropolysaccharides composed of a variable number of repeating disaccharide units. Each disaccharide consists of one hexosamine,
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_16, © Springer Science+Business Media New York 2015
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Nicola Volpi and Francesca Maccari D-galactosamine or D-glucosamine and one uronic acid, Dglucuronic acid or L-iduronic acid (IdoA) or neutral hexose, D-galactose in keratan sulfate. According to the type of the monosaccharide units and the glycosidic bonds between them, GAGs can be divided into four main categories: (1) hyaluronic acid or hyaluronan (HA), (2) chondroitin sulfate (CS) and dermatan sulfate (DS), (3) heparan sulfate (HS) and heparin (Hep), and (4) keratan sulfate (KS) (Fig. 1). Separation of GAGs by electrophoresis is routine in many laboratories and their characterization to the microgram level forms an integral part of biochemical research, especially with respect to obtaining information from unknown purified polysaccharides.
Fig. 1 Structures of disaccharides forming GAGs. Major modifications for each structure are illustrated (R = H or SO3−) but minor variations are possible
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This is more important for natural (macro) molecules produced by extraction and purification from different animal tissues having several fundamental biological activities, as well as pharmacological properties, making them important drugs for use in clinical and pharmaceutical fields [1–5]. Cellulose acetate [6], nitrocellulose membrane (NC) [7], agarose-gel [8], and polyacrylamide-gel [9, 10] electrophoretic techniques are generally utilized for GAGs isolation and separation and for qualitative and quantitative analyses of their mixtures or single species. However, agarose-gel electrophoresis permits the separation and identification of several GAG species, such as slow (SM heparin) and fast moving heparin (FM heparin) or heparan sulfate, dermatan sulfate, and chondroitin sulfate [8, 11]. GAGs are strongly hydrophilic and negatively charged macromolecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. As a consequence, membranes have been derivatized with cationic detergents to make them hydrophilic and positively charged, like cetylpyridinium chloride (CPC)-treated NC membranes used in this protocol. After their electrophoretic separation, several intact GAGs with high molecular mass, such as HA, CS, highly sulfated CS, DS, HS, Hep, and its two components, FMHep and SMHep species, were transferred on NC membranes treated with a cationic detergent, CPC [12]. Quantitative analysis was performed after visualization of bands by cationic dyes, the recovery of single molecules released from membrane was also examined [12], and the direct and specific recognition of these polysaccharides by antibodies on CPC-treated NC supports has also been described [13].
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Materials 1. High purity agarose (see Note 1). 2. Barium acetate and NC membranes 0.45 mm, binding capacity of 80–100 μg/cm2. 3. 1,2-Diaminopropane (PDA) and cresol red. 4. Cetylpyridinium chloride (CPC). 5. Toluidine blue, alcian blue, and Whatman 3 MM paper. 6. All the other reagents should be of analytical grade.
2.1 Glycosaminoglycans
1. Different GAGs to be used as standard may be purchased from Sigma-Aldrich (http://www.sigmaaldrich.com) or other specialized companies. 2. Extraction and purification protocols for various GAGs are available in specific scientific articles and monographs [3, 5, 14–21].
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1. 40 mM barium acetate buffer, pH 5.8 with 1 M acetic acid. Store at 4 °C. 2. 50 mM 1,2-diaminopropane (PDA) buffer: Buffered at pH 9 with glacial acetic acid. Store at 4 °C. 3. Cresol red solution: Dissolve 10 mg of the dye in 100 mL of distilled water (final concentration of 0.1 mg/mL). Store at 4 °C.
2.3 Blotting on Membranes
1. 1 % CPC in 30 % 2-propanol. This solution should be always freshly prepared. 2. 150 mM NaCl. 3. Transfer buffer: 100 mM Tris-acetate buffer pH 7.3. Store at 4 °C.
2.4 Membrane Staining
1. Alcian blue solution: Dissolve 50 mg of the dye in 1 mL 8 M guanidine and 19 mL of 18 mM sulfuric acid-0.25 % Triton X-100. This staining reagent is always freshly prepared. 2. Reversible staining is always freshly prepared with toluidine blue. 20 mg toluidine blue is dissolved in 100 mL of 3 % acetic acid.
3
Methods
3.1 Agarose-Gel Electrophoresis of Glycosaminoglycans
1. Prepare 0.5 % agarose solution in 40 mM barium acetate buffer (see Note 2). Heat the solution in a microwave oven or on a stirrer mixer, mixing continuously until the agarose completely dissolves and a clear solution is obtained. Do not allow the solution to boil. 2. Thoroughly clean a single glass plate of 7 × 8 cm (of approx. 2 mm thickness) with alcohol. After drying with paper, place it in a holder of approx. 8 × 10 cm and pour the warm agarose solution (see Notes 3 and 4). 3. Prior to electrophoresis, leave the gel at room temperature (RT) for approx. 30 min. Cut the gel by the side of the glass plate and put it with the same glass plate on a grid made of 1 × 1 cm squares. Make four small wells by using a flat chisel of approx. 5 mm, taking care to leave approx. 5 mm between each well (see Note 5). Make the wells approx. 2 cm from the edge of the gel. 4. 10 μL of GAGs standard or samples (see Note 6) may be layered by micropipets into the wells. 5. The electrophoretic run is performed in 50 mM PDA for 150 min at 50 mA by using a Pharmacia Multiphor II electrophoretic cell instrument (see Note 7).
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1. Cut NC membranes in portions of 7 × 8 cm. 2. Wet the membrane in freshly prepared 1 % CPC in 30 % 2-propanol for 5 min. 6 mL of CPC solution should be used. Shake the membrane manually. 3. After wetting, add 50 mL of 150 mM NaCl and incubate the membrane on a shaker for 15 min. 4. CPC-derivatized membrane is rinsed several times in 150 mM NaCl (see Note 8) and then equilibrated with continuous shaking in the same NaCl solution until blotting is performed. 5. After agarose-gel electrophoresis, carefully remove the gel from the glass plate. 6. Prepare the blotting sandwich by assembling a Whatman 3 MM paper immersed in the buffer reservoir (see Note 9). Carefully place the agarose gel on the 3 MM paper with the wells located parallel to the two buffer reservoirs. The detergent-treated NC membrane is then laid on top of the gel (see Note 10). Three further wetted filter papers, two wetted sponges, and 5 cm of absorbent paper tissue are carefully laid on top of the NC membrane, ensuring that no bubbles are trapped in the resulting sandwich. The blotting sandwich is stabilized by putting a 500 g weight on the top. 7. The capillary blotting is performed overnight at RT.
3.3 Staining Procedures
1. Irreversible staining is performed by means of alcian blue. After 2 h staining, membrane is destained by rinsing in 150 mM NaCl until background staining disappears (see Note 11). An example of the results produced is shown in Fig. 2a.
Fig. 2 (a) Decreasing amounts (a: 7.5 μg, b: 5.0 μg, c: 2.5 μg, d: 0.1 μg) of mixtures of GAGs composed of (1) slow moving heparin, (2) fast moving heparin, (3) dermatan sulfate, and (4) chondroitin sulfate electrophoretically separated by agarose-gel and blotted on CPC-treated NC membrane. The membrane was stained with alcian blue. (b) Quantitation of immobilized sulfated and nonsulfated GAGs on CPC-treated NC membranes after agarose-gel electrophoresis, capillary blotting, staining with alcian blue, destaining, and densitometric analysis. Hep heparin, SM Hep slow moving heparin, FM Hep fast moving heparin, DS dermatan sulfate, CSi highly sulfated chondroitin sulfate, HS heparan sulfate, CSb chondroitin sulfate, HA hyaluronic acid, K4 bacterial polysaccharide K4, K4 Defructosylated defructosylated bacterial polysaccharide K4. Reprinted with permission
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2. Irreversible staining with alcian blue can be used for quantitative studies with a high detection sensitivity (see below). Quantitative analysis of GAGs may be performed with a densitometer connected to a computer by using an image processing and analysis software. The wet membranes should be scanned in the RGB mode and saved in gray scale. An example of the results produced is shown in Fig. 2b. 3. GAGs separated by agarose-gel electrophoresis and transferred to a membrane can also be detected by toluidine blue staining (see Note 12). Reversible staining may be obtained by treating membranes with toluidine blue solution for 5 min. The membrane is rinsed for 30–60 s in 3 % acetic acid in the presence of 0.1 % CPC to remove the excess stain, and further utilized (see Notes 13 and 14).
4
Notes 1. Agarose should be of very high quality, suitable to run high resolution gels (possibly certified for molecular biology, ideal for the separation of small DNA fragments). Agarose from Sigma-Aldrich or from Bio-Rad is suitable. 2. The volume of the agarose solution is strictly related to the dimension of the gel. For a gel of 7 × 8 cm with a thickness of about 4–5 mm, a volume of 50 mL (250 mg agarose) is advisable. 3. Carefully eliminate possible air bubbles in the warm solution, and allow the agarose solution to convert into a gel at RT for about 30–60 min. The gel may be stored at 4 °C for approx. 4–5 days after covering it with a plastic sheet. 4. It is very important to consider that a gel having a thickness lower than about 4–5 mm does not permit the layering of the samples. On the contrary, a gel with a greater thickness requires greater migration times. 5. Carefully dry the wells by using little pieces of Whatman 3 MM paper of approx. 5 × 20 mm. 6. GAGs standard should be prepared at a concentration of 0.5 mg/mL in distilled water with a final absolute amount of 5 μg loaded on the gel. Extracted GAGs from different matrices should be quantitatively evaluated by means of known assays [1, 13–15, 17] before performing the electrophoretic separation. The optimum concentration range of unknown purified GAGs loaded on the gel should be from 2 to 8 μg. 7. Due to the possible variability of the electrophoretic conditions, 2 μL of cresol red solution (0.1 mg/mL) should be added to each standard or sample solution (10 μL) in order to
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make a more accurate evaluation of the electrophoretic migration. A good migration time and electrophoretic separation are obtained at a cresol red migration of approx. 20–25 mm. 8. Derivatized membranes should be rinsed several times in 150 mM NaCl with vigorous shaking until no foaming is observed. This step permits the complete removal of excess CPC. 9. The Whatman 3 MM is immersed in the two buffer reservoirs permitting the migration of the buffer from the tank to the top side of the blotting sandwich. As a consequence, make sure that sufficient buffer volume for a complete GAGs migration (approx. 1 L) is available. 10. Carefully remove possible air bubbles entrapped between the gel and the membrane by using a little glass pestle. 11. It is very important to optimize the irreversible staining period to obtain a good band staining against a clear background. Under the experimental conditions described, an optimum staining time would be 2 h. Furthermore, use several changes of the destaining solution to produce the best results. 12. The sensitivity of staining with toluidine blue is about 10–15 times lower than that with alcian blue (an example is illustrated in Fig. 3), but if the membrane is then destained, the same lanes can be used for immunological detection or other applications.
Fig. 3 Quantitation of immobilized sulfated and nonsulfated glycosaminoglycans on CPC-treated NC membranes after agarose-gel electrophoresis, capillary blotting, staining with toluidine blue, destaining, and densitometric analysis. Hep heparin, SM Hep slow moving heparin, FM Hep fast moving heparin, DS dermatan sulfate, CSi highly sulfated chondroitin sulfate, HS heparan sulfate, CSb chondroitin sulfate, HA hyaluronic acid, K4 bacterial polysaccharide K4, K4 Defructosylated defructosylated bacterial polysaccharide K4. Reprinted with permission
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