ANALYTICAL

BIOCHEMISTRY

1%,238-242

(1991)

Optimization of an Alcian Blue Dot-Blot Assay for the Detection of Glycosaminoglycans and Proteoglycans Luc Buee,**-f

Noel

J. Boyle,*

Libang

Zhang,*

Andre

Delacourte,?

and Howard

M. Fillit*$

*The Ritter Department of Geriatrics, The Mount Sinai Medical Center, One G. L. Levy Place, New York, New York 10029; TUnite 156 INSERM, Laboratoire de Neurosciences, Place de Verdun, 59045 Lille Cedex France; and $The Fishberg Center for Neurobiology, The Mount Sinai Medical Center, One G. L. Levy Place, New York, New York 10029.

Received

November

2,199O

A modification of a method for the detection of proteoglycans on positively charged nylon was adapted for use in a dot-blot assay and improved. Different dot-blot membranes were tested and variations in the Alcian blue staining solution (including pH, critical electrolyte concentration, and ionic strength) were explored. With modifications, we were able to eliminate interferences by other polyanions such as DNA or proteins. We were able to detect glycosaminoglycans and proteoglycans down to the 10 ng range. Furthermore, our assay is compatible with high concentration of urea (up to 7 M) used in classic proteoglycans extraction methods and is a useful tool to monitor the isolation of proteoglycans by anion exchange and gel filtration chromatography. It is technically easier, faster, more sensitive, and more specific than previously published methods and can be adaptated as a quantitative assay using a scanning densitometer with a linear range of detection from 10 to 100 ng of glycosaminoglycans. o 1991 Academic PWS, IIIC.

Proteoglycans (PGs)’ consist of a protein core and covalently linked glycosaminoglycans (GAGS) which are linear polysaccharides (mainly hexuronic acid and hexosamine) containing sulfate groups. Most assays used to detect proteoglycans are based on the detection of carbohydrate content such as uranic acid (l), galactosamine, or glucosamine (2). Cationic dyes such as Al-

1 Abbreviations used: PGs, proteoglycans; GAGS, glycosaminoglycans; PVDF, polyvinylidene difluoride; CSA, chondroitin sulfate A, CSC, chondroitin sulfate C, DS, dermatin sulfate; HS, heparan sulfate; Hp, heparin; HA, hyaluronic acid; KS, keratan sulfate; BCA, hicinchoninic acid; BSA, bovine serum albumin; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]propanesulfonic acid, Gua-HCI, guanidine hydrochloride; HSPG, heparan sulfate proteoglycan; CEC, critical electrolyte concentration.

cian blue (3), toluidine blue (4), or 1,9dimethylmethylene blue (5) which stain polyanions have been used in many assays for the detection of PGs, including densitometric scanning of cellulose acetate electrophoregrams (6) and quantitation of the color by spectrophotometry (7-9). The limit of detection for these assays is about 2 pug/ml. However, the methodology employed for the extraction of PGs from various tissues generally requires high concentrations of salts or urea. Proteins and nucleic acids are also commonly encountered during PGs extraction procedures. All of these interfere with previously described assays. Recently, Heimer and Sampson (10) demonstrated the advantage of detecting PGs on positively charged nylon (10). We show here that it is possible to use a dot-blot method to detect PGs and/or GAGS in urea buffer and in the presence of other highly negatively charged macromolecules. Moreover, the sensitivity of this assay has been optimized by varying the concentrations of Alcian blue, MgCl,, pH, ionic strength, and the type of membranes. The best combination of these factors allowed us to detect GAGS and PGs down to a lo-ng range. MATERIALS

AND

METHODS

Materials. Immobilon-N and Immobilon-P membranes (polyvinylidene difluoride-based membranes (PVDF), pore size: 0.45 wrn) were purchased from Millipore Corp. (Bedford, MA). Nitrocellulose membrane filters (BA 85, 0.45 pm) and positively charged Nytran 66 sheets (0.2 pm) were purchased from Schleicher and Schuell (Keene, NH). Zetabind transfer membranes (0.45 pm) were purchased from CUNO Division (Meriden, CT). Zeta-Probe blotting membranes (0.45 pm) and DEAE-cellulose papers were purchased from BioRad Laboratories (Richmond, CA). The following GAG standards were obtained from Sigma Chemical Co. (St.

238 All

0003-2697/91$3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.

OPTIMIZATION

OF

Louis, MO): chondroitin sulfate A (CSA) from bovine trachea, chondroitin sulfate C (CSC) from shark cartilage, dermatan sulfate (DS) from porcine skin, heparan sulfate (HS) from bovine kidney, heparin (Hp) from porcine intestinal mucosa, hyaluronic acid (HA) from human umbilical cord, and keratan sulfate (KS) from bovine cornea. All GAG preparations were shown protein free using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Bovine serum albumin (BSA) and deoxyribonucleic acid (DNA) were obtained from Sigma. Alcian blue 8 GX was also purchased from Sigma. All the other chemicals used were of analytical grade. Buffers. The buffers were as follows: phosphatebuffered saline (PBS): 0.05 M NaCl, 0.012 M Na,HPO,, and 0.003 M NaH,PO,, pH 7.2; urea buffer: 10 mM bisTris, pH 5.8, 50 mM Na acetate, 7 M urea, and 0.05% (w/v) Chaps; and guanidine hydrochloride (Gua-HCl) buffer: 4 M Guanidine-HCl, 50 mM Na acetate, pH 5.8, 0.5% (w/v) CHAPS. PG preparations. Purified vascular heparan sulfate proteoglycan (HSPG) from bovine kidney was obtained by methods previously described and has been previously characterized with regard to its purity and composition (11). Briefly, glomeruli were obtained from bovine kidney by a sieving method. After a hypotonic lysis, a 4 M Gua-HCl extraction, and stepwise chromatography on a DEAE-Sepharose CL-6B (Pharmacia, Piscataway, NJ) column, PGs were further purified by gel filtration on a Sepharose CL-4B (Pharmacia) column in urea buffer. The retarded fraction (I(,, = 0.48) contained primarily heparan sulfate proteoglycan (HSPG). This material was used for the dot-blot assay. Monoclonal antibodies against HSPG. Characterization and specificity of the monoclonal antibodies to HSPG from bovine kidney (4F2 and 7E12) have been described previously (12). Dot blotting. Adsorptive immobilization of 50-~1 samples of GAGS or PGs dissolved in a loading buffer was done in a Bio-Dot microfiltration apparatus (BioRad Laboratories) by gravity flow. After passive filtration, 200 ~1 of PBS was added under a vacuum to each well to ensure that all the sample and the salts are removed from the drain ports underneath the membrane. The membrane was removed from the Bio-Dot apparatus and placed in a solution of PBS with 0.5% Tween 20 for 30 min. Then it was stained with Alcian blue for 20 min. Alcian blue staining solution. The components of the staining solution were Alcian blue, acetic acid or Na acetate, NaCl, and MgCl,. The optimum concentration of each component was investigated. Two main compositions of the staining solution were used: (A) 0.2% (w/v) Alcian blue; 0.05 M Na acetate, pH 5.7; 0.05 M NaCl, and 0.05 M MgCI,; (B) 0.2% (w/v) Alcian blue; acetic acid, pH 2.7; 0.05 M NaCl; and 0.3 M MgCl,.

ALCIAN

BLUE

ASSAY

239

Quantitation of Alcian blue staining by densitometry. Dried membranes were scanned in order to quantify the staining using an LKB Pharmacia Ultroscan XL laser densitometer. RESULTS

Optimization of the Alcian Blue Staining Solution Initial experiments to establish the optimal conditions of staining were made on Zetabind membranes with HS and CSC dissolved in PBS buffer. The percentage (w/v) of Alcian blue was varied from 0.01 to 1% at two different pH (2.7 and 5.7) in the presence of 0.05 M MgCl,. At pH 2.7, the staining for CSC was maximum at 0.1% Alcian blue and then decreased at higher concentrations of Alcian blue. There was no interference, when compared at same concentrations, by other polyanions (DNA or BSA) or even with HA at pH 2.7. At pH 5.7, the staining of CSC and HS increased with the percentage of Alcian blue. However, with a percentage of Alcian blue greater than 0.2%, we observed high interferences with DNA and BSA, and high background staining. Thus 0.2% Alcian blue was the optimum concentration to stain HS and CSC and to avoid interferences. Effect of MgCl, Concentration, pH, and Ionic Strength (NaCl Concentration) After initial experiments, we particularly studied the Alcian blue staining at pH 2.7 and 5.7 for two concentrations of MgCl, (0.05 M and 0.3 M). As previously reported, HS and CSC were stained with the Alcian blue solution at low concentrations of MgCl, while only CS was stained at higher concentrations (3,13). At pH 2.7 and 0.3 M MgCl,, CS staining was enhanced while HS staining was decreased (Fig. 1). At pH 5.7, with a concentration of 0.05 M MgCl,, both HS and CSC were stained with a high sensitivity, but a light background was also observed with other polyanions. Under these conditions, it was possible to enhance the sensitivity and specificity of PGs/GAGs staining by increasing the ionic strength of the staining solution. NaCl concentration was increased from 0 to 0.6 M. Up to 0.05 M NaCl, we observed an increase of the GAGS and PGs staining while the staining of the other polyanions was decreased. With a concentration of NaCl more than 0.1 M, the staining of GAGS and PGs was decreased (data not shown). Binding of GAGS and PGs to Different Blotting Membranes To test different blotting membranes, the following staining solution was used: 0.2% Alcian blue; 0.05 M Na acetate, pH 5.7; 0.05 M MgCl,; and 0.05 M NaCl. We chose to work with seven representative mem-

240

BUEE

12345678 A&s, B 1 c D ! E F G

e,e ‘a e 1

:

HS

I

cs

FIG. 1. Effect of pH and of MgClx on Alcian blue staining of heparan sulfate (HS) and chondroitin sulfate C (CS). Each GAG was diluted from A to G (respectively, 250, 125,62.5,31.2, 15.6, 7.8, and 0 ng) in PBS and then loaded on Zetabind. Four Alcian blue staining solutions were studied: Lanes 1,5: 0.2% Alcian blue and 0.05 M MgCI, in acetic acid, pH 2.7. Lanes 2,6: 0.2% Alcian blue and 0.3 M MgCl, in acetic acid, pH 2.7. Lanes 3, 7: 0.2% Alcian blue and 0.05 M MgCl, in 0.05 M Na acetate, pH 5.7. Lanes 4, 8: 0.2% Alcian blue and 0.3 M MgClz in 0.05 M Na acetate, pH 5.7. CS staining is better than HS staining at low pH and high concentration of MgCI, (0.3 M). Optimal staining of both GAGS was obtained at pH 5.7 and low concentration of M&l, (0.05 M).

ET

AL.

Gel Filtration Assay

Monitoring

by the Al&n

Blue Dot-Blot

In order to demonstrate the efficiency and practicality of this assay, an extraction of HSPG from bovine kidney was monitored by both the Alcian blue dot-blot assay and by an immuno-dot-blot assay with antibodies against HSPG (12). The staining solution of Alcian blue also detected other PGs in peak A (11) of the gel filtration on Sepharose CL-4B and also GAGS and/or proteolytic fragments of PGs in peak C (Fig. 4). However, by immuno-dot-blot employing monoclonal antibody 4F2, HSPG protein core was detected only in peak B. DISCUSSION

Alcian blue is commonly used to stain GAGS and PGs. Nevertheless, since the works of Scott which were primarily adapted for histology (3,14), exploration of the different factors involved in the binding of this dye has not been performed. Furthermore, new binding membranes have appeared but only a few have been tested

1 2 3 4 5 6 7 8 9 10 1112

branes (Immobilon-N, Immobilon-P, nitrocellulose, Nytran, Zetabind, DEAE-cellulose, and Zeta-probe) (Fig. 2). Only PGs but not free GAGS bound to Immobilon-P and nitrocellulose membranes. GAGS also did not bind to Nytran. DEAE-cellulose did not give reliable results. Two nylon membranes (Zetabind and Zetaprobe) and one PVDF based membrane (Immobilon-N) gave excellent results with both GAGS and PGs, even with low dilutions of polyanions. Loading Buffers Different buffers were used to dissolve GAGS or PGs: PBS buffer, urea buffer, Gua-HCl buffer, and CsCl solution. Only GAGs/PGs dissolved in high concentration of CsCl solutions (1.2 g/g of solution) did not give any staining. Ionic buffers such as PBS and Gua-HCl buffers gave good staining but urea buffer showed the best results. Quuntitation

on Zetabind Membranes by Densitometry

Dilutions of CSA, CSC, DS, HS, Hp, KS, and HSPG were made in urea buffer and loaded on Zetabind membranes. After Alcian blue staining (0.2% Alcian blue0.05 M Na acetate, pH 5.7-0.05 M MgCl,-0.05 M NaCl), the dried sheets were scanned. A linear range (lo-100 ng) was established between the amount of GAGS loaded on dot-blot and the staining intensity (Fig. 3a). The linear range between the amount of KS loaded and the staining intensity was from 100 to 500 ng. A linear range (20-200 ng) was also found between the amount of HSPG and the staining intensity (Figure 3b).

A B

Immobilon-N 1 ”

e d a i

a**

b

Immobilon-P A B Nitrocellulose A B Nytran 66 A B Zetabind *a*

A B

eer Zeta-Probe

A B

e * 5 *e

FIG. 2. Alcian blue staining of polyanions bound to different blotting membranes, Six molecules at four different dilutions in PBS (1.25 Fg, 500, 250, and 50 ng) were tested: (Al-A4) Heparan sulfate (HS), (A5-A8) chondroitin sulfate C (CSC), (A9-A12) DNA, (Bl-B4) hyaluronic acid (HA), (B5-B8) bovine serum albumin (BSA), and (B9-B12) HSPG. Each membrane was then immersed in the Alcian blue solution (0.2% Alcian blue, 0.05 M MgCl,, 0.05 M NaCl, 0.05 M Na acetate, pH 5.7). Only three membranes (Immobilon-N, Zetabind, and Zeta-probe) showed adequate binding of both GAGS and PGs and significant staining by Alcian blue without interference by other polyanions.

OPTIMIZATION

z g

0.0-I 0

c

8

8

0

8

1

20

40

60

80

100

120

Glycosaminoglycan

v1

.?t

‘“1

OF

(ng)

b

ALCIAN

BLUE

wards sulfated GAGS or PGs if the salt concentration is suitably chosen. We investigated different pH and different concentrations of MgCl, in the dot-blot assay. At pH 2.7, presumably most negative groups (carboxylate, phosphate, or sulfate) lost their charges, but CSC staining was slightly enhanced while HS staining was decreased. These differences may be due to the fact that CSC has somewhat more negative sulfate charges than HS (16) and was probably better able to bind the dye in presence of high concentrations of MgCl, (0.3 and 0.6 M). In addition, there may be conformational differences between the two GAGS. At pH 5.7 and 0.05 M MgCl,, all molecules with numerous sulfate groups are stained. However, we observed a light background due to nonspecific binding. It was possible to clear this background by increasing the ionic strength to 0.05 M

Relative Units 40 Heparan

80 Sulfate

120 Proteoglycan

160

241

ASSAY

Absorbance (280nm)

200

(ng)

FIG. 3. Quantitation by densitometry of Alcian blue staining binding to materials loaded on Zetabind membranes. Materials (O-250 ng) were diluted in urea buffer and then dot-blotted onto Zetabind membranes. After Alcian blue staining (0.2% Alcian blue, 0.05 M MgCl,, 0.05 M NaCl, 0.05 M Na acetate, pH 5.7), the dried sheets were scanned to quantify the staining using an LKB Ultroscan XL Laser densitometer. (a) A linear range (lo-100 ng) between the amount of the five glycosaminoglycans loaded (CSA 0, CSC A, DS 0, HS n , and Hp Cl) and the staining intensity was established. (b) A linear range (20-200 ng) between the amount of heparan sulfate proteoglycan loaded (HSPG 0) and the staining intensity was established.

for binding capacity of GAGs/PGs (10,15). In this study, we optimized the conditions of Alcian blue staining already established by others and we tested new membrane supports. The optimum percentage of Alcian blue in the staining solution was established: 0.2% (w/v). Indeed, a higher concentration (>0.2%) of Alcian blue enhanced nonspecific binding more than specific staining of GAGS and PGs. A lower concentration (~0.2%) of Alcian blue reduced sensitivity. Next, we studied the best buffer conditions for binding of Alcian blue. The binding of a polyanion to a dye depends primarily on its number of negative groups (carboxylate, phosphate, or sulfate). Furthermore, a competition occurs between the dye and buffer cation(s) for these anionic sites. Each negative group has a different affinity for the dye and for a given inorganic cation. The sulfate ester group is the last to give up its dye in presence of MgCl,. The concentration where a given inorganic cation is high enough to displace the dye is called the critical electrolyte concentration (CEC) (14). Thus, it should be possible to produce a staining solution which is directed to-

Fraction

Alcian

number

blue

1 2 3 4 5 6 7 8 9 10 1112

. Anti-HSPC, 1 2 3 4 5 6 7 8 9 10 1112

FIG. 4. Gel filtration of proteoglycans extracted from bovine kidney on Sepharose CL-4B monitored by the Alcian blue dot-blot assay and by immuno-dot-blot assay. Fifty microliters of each &ml fraction was dot-blotted onto Zetabind membrane. After 1 h, the Zetabind membrane was washed for 30 min in PBS-Tween. The membrane was then immersed in the staining solution (0.2% Alcian blue, 0.05 M MgCl,, 0.05 M NaCl, 0.05 M Na acetate, pH 5.7) for 15 min. The membrane was finally washed in the destaining solution. Fifty microliters of each fraction was also dot-blotted and then incubated with a monoclonal antibody against bovine kidney heparan sulfate proteoglycan. Row I (fractions 1 to 12), row II (fractions 13 to 24), row III (fractions 25 to 36), and row IV (fractions 37 to 48). The Alcian blue dot-blot assay detected proteoglycans and/or glycosaminoglycans in all peaks whereas the monoclonal antibody detected only the vascular heparan sulfate proteoglycan protein core (fractions II-4 to 111-g).

242

BUEE

NaCl concentration. This balance between an increase of the ionic strength and MgCl, concentration allowed staining of both GAGS or PCs (Figs. 2 and 3) with almost no interferences by other polyanions including HA. The binding capacity of dot-blot membranes is an important factor in the detection of GAGs/PGs. Immobilon-P and nitrocellulose are two membranes which show good protein binding (17). PGs bound to them well but GAGS binding was less than optimal. We also studied positively charged membranes (Immobilon-N, Nytran, Zetabind, DEAE-cellulose, and Zeta-probe) which allow ionic interactions (l&19). Nytran 66, despite a smaller pore size (0.22 km) than other membranes (0.45 pm) did not bind GAGS at low concentrations. This confirmed the report of Heimer et al. (15) who were not able to detect by lz51-cationized cytochrome c GAGS immobilized on Nytran 66 when the concentration was less than 100 ng. GAGs/PGs dot-blotted on DEAE-cellulose could not be readily stained by the Alcian blue solution due to the retention of the dye to this support and its low wet strength. Only three positively charged membranes were really efficient (Immobilon-N, Zetabind, and Zetaprobe). We chose to work with Zetabind which does not require any preparative steps such as an initial methanol bath for PVDF membranes. The high binding of GAGs/PGs to these membranes could be explained by an interaction between the negative charges of GAGS chains and the positive charges of the membranes. Since KS showed a lighter detection on a per dry weight basis but contains no uranic acid and approximately the same amount of sulfate groups (16) as other GAGS, it is likely that GAGS binding to membranes involves both the carboxyl group of uranic acid as well as the sulfate groups. The initial conditions of Alcian blue staining established with CSC and HS were adapted to the staining of other GAGs/PGs (Figs. 3 and 4). Bovine kidney HSPG staining showed a wider linear range than HS chains extracted from bovine kidney. The apparent differences in sensitivity of the assay for the detection HS and HSPG are probably related to the amount of GAGS in each preparation, since HSPG is only approximately 30% (w/w) GAGS. In addition, the presence of protein in HSPG and not in HS likely alters the membrane binding and the dye binding properties of these molecules. Alcian blue staining sensitivity for GAGs/PGs was maximum when urea buffer was used as loading buffer. Other buffers such as PBS and Gua-HCl result in lower assay sensitivity. It seems likely that ionic interactions of the salts in PBS and Gua-HCl buffers reduced the binding of GAGs/PGs to the membrane.

ET

AL.

The optimal assay described in our study is useful for the quantitation of GAGs/PGs at very low dilutions without requiring radioactive labeling. This assay represents a practical and sensitive tool for the detection of PGs and GAGS even in the presence of harsh extraction and chromatographic buffers commonly employed in PGs biochemistry. It is an easy way to quantify GAGS and PGs dissolved in urea buffer in nanogram concentrations with greater sensitivity and specificity than previously described calorimetric methods. ACKNOWLEDGMENTS This work was supported by grants from NIH NIAID-AI24876-01, from NIH NIA P50-AG05138-07, and from the Florence J. Gould Foundation. L.B. is a visiting scientist of the Florence J. Gould foundation and is also sponsored by the MRT (Research and Technology ministry, France).

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