Iozzo Renato V. Proteoglycan Protocols

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150 Dodge and Heimer

and RNA (5). Improvement of this technique, originally done in tube gels, led inevita-

bly to the use of slab gels (6) and also to improvement in sample preparation through

use of SDS and reduction (6) or SDS only (7). These modifications facilitated the

analysis of tissue extracts and biologic fluids without prior fractionation. Originally

the detection of proteoglycans was accomplished with the basic thiazine dye, toluidine

blue. Increased sensitivity of the detection of proteoglycans was achieved by tech-

niques such as biosynthetic labeling with

(6) transblotting to positively charged

Nylon membranes followed by staining with Alcian blue (7), and transblotting to

unmodified Nylon and staining with the highly positively charged

125

I-cytochrome c

(8). The detection achieved with Alcian blue staining of proteoglycans on positively

charged Nylon-66 is nearly 100 times more sensitive than staining with toluidine blue

(7). With the availability of monoclonal and polyclonal antibodies specific for diverse

proteoglycans (the core proteins and associated GAGs), standard Western blotting tech-

niques could be utilized on proteoglycans transblotted from composite gels. Table 1

contains a partial list of antibodies useful for the characterization of a wide range of

known proteoglycans.

An interesting application of Western blotting involves sequential testing of a

single blot with multiple antibodies. Antibodies bound to the proteoglycan antigen

transblotted to nitrocellulose can be removed by exposure to sodium thiocyanate

without materially affecting the binding of the immobilized material. The blot can

then be probed again by another antibody. Densitometry scans of each blot can then be

superimposed on one another, and by visual inspection this procedure can aid in

resolving closely migrating species and also in determining the purity of a proteoglycan

isolate or a standard (9).

Table 1

Monoclonal and Polyclonal Antibodies Useful in Characterizing Proteoglycans

Antigen Name Polyclonal/mAb Notes Reference

Perlecan 7B5 mAb Laminin-like region 19

Versican 2B1 mAb 20

Keratan sulfate AN9P1 mAb Bound to aggrecan 21

Aggrecan BE123 mAb After c-ABC-treatment 22

Aggrecan, N-terminal BC3 mAb After aggrecanase exposure 23

Aggrecan, C-terminal BC4 mAb After aggrecanase exposure 23

Aggrecan IC6 mAb HA-binding domain 24

Chondroitin sulfate CS56 mAb Bound chondroitin 4 or 6 sulfate 25

Chondroitin-4 sulfate 2B6 mAb After c-ABC digestion 26

Chondroitin-6 sulfate 3B3 mab After c-ABC digestion 27

Unsulfated dissacharide 1B5 mAb After c-ABC digestion 28

Biglycan LF15 Poly N-terminus 29

Decorin LF30 Poly N-terminus 29

Gel Electrophoresis and Immunoblotting 151

Results obtained with agarose/acrylamide gel electrophoresis have greatly enhanced

our knowledge of proteoglycan biochemistry, synthesis, and degradation. First, avoid-

ing time-consuming preparative steps has allowed the direct analysis of biologic speci-

mens and tissue extracts. In general, such analyses have been useful in the simultaneous

detection of proteoglycans with relatively large core proteins conjointly with detec-

tion of proteoglycans of smaller size. While large and small proteoglycans are easily

separable, identification within each group can best be made by specific antibodies.

The chief difficulty with separating proteoglycans with large core proteins is the pres-

ence of multiple glycosaminoglycan side chains and that the side chains often show

size heterogeneity, which then affect overall mobility and electroendosmosis. An

example of the number of glycosaminoglycan side chains affecting the mobility is illus-

trated when biglycan with its two glycosaminoglycan side chains has to be distinguished

from decorin, with its single side chain. The core proteins of these proteoglycans have

a rather similar molecular weight, although the presence of one additional (two)

dermatan/chondroitin sulfate side chain biglycan results in biglycan having a molecu-

lar weight of approximately 100,000 daltons and decorin, with one such chain, only

70,000 daltons. One would assume, therefore, that this pair would be readily sepa-

rable. Figure 1 is an example of the power of using composite gels and Western blot-

ting and demonstrates this by using two different antibodies, CS-56 (reactive with

intact chondroitin 4- and 6-sulfate chains) and 2-B-6 (reactive with terminal

dissacharides remaining on the core protein of proteoglycans after digestion with

chondroitinase ABC (26). One can see in Fig. 1A that, when using monoclonal CS-56,

Fig. 1. Demonstration of different chondroitin/dermatan sulfate proteoglycans in myocar-

dial extracts. Western blots (A, B) prepared from samples of primate myocardial extracts fol-

lowing composite agarose-acrylamide gel electrophoresis were incubated with a 1:200 dilution

of the monoclonal CS-56 (ICN) (which reacts with intact chondroitin-4 and -6 sulfate chains)

(A) or a 1:200 dilution of the monoclonal 2-B-6 (which reacts with terminal unsulfated disac-

charides on chondroitinase ABC digestion) (25). Lane 1 is the extract of the primate myocar-

dium, lane 2 is a standard of 50 ng of bovine aggrecan from articular cartilage. (B) Western of

duplicate samples that were exposed overnight at 37°C to 0.1 unit of chondroitinase ABC (ICN)

in 5 mL of 0.2 M Tris buffer, pH 8.0, before being incubated with monoclonal 2-B-6. This

demonstrates the presence of two clearly defined proteoglycan bands in the mycardial extract

(lane 1), discernable only after chondroitinase digestion.

152 Dodge and Heimer

one band was identified in the myocardial extract (see lane 1) that migrated ahead of

the aggrecan standard (see lane 2). When an identically prepared blot (see Fig. 1B)

was incubated first with chondroitinase ABC, then with a dilution of the monoclonal

2-B-6, two bands became evident in the myocardial extract, indicating the presence

of two distinct species of chondroitin-4 sulfate-containing proteoglycans, presum-

ably decorin and bigylcan. The precise identity of these proteoglycans would best be

confirmed with specific decorin and biglycan antibodies (reactive with the core pro-

teins). Shown in Fig. 2, using specific antibodies for the biglycan (see lane 1) and

the decorin (see lane 2) core protein, one would be unable to distinguish them by

just using monoclonal antibodies directed to the common dermatan/chondroitin sul-

fate side chain (10,11). It is remarkable that although biglycan and decorin have a

similar mobility in composite gels, biglycan shows a greater polydispersity due to

its more complex orientation and altered electroendosmotic effects.

Surprisingly, a search of the literature reveals that agarose/acrylamide gel electro-

phoresis has not been as widely used as this powerful method (see Note 1) would deserve.

Historically, the major areas where agarose/acrylamide gel electrophoresis has been use-

ful has been in the analysis of guanidine-HCl extracts of tissues, such as cartilage, and

tendon. We summarize here in chronological order some of these findings.

Two types of aggregating proteoglycans were found by zonal rate centrifugation

of guanidine-HCl extracts of bovine cartilage, one of Mr 3.5 × 10

and the other of

Mr 1.3 × 10

. The former did not contain a well-defined keratan sulfate-rich domain;

the latter, however, contained the same percentage of hexosamine, and perversely

the smaller species contained 20% more protein than the larger. Agarose/acrylamide

gel electrophoresis showed one band for each proteoglycan and that the larger one had

Fig. 2. Example of a composite agarose/acrylamide gel/Western blot that demonstrates the

presence of bigylcan and decorin found in an extract of myocardium. Primate myocardium was

extracted with 4 M guanidine-HCl, dialyzed against 6 M urea and aliquots electrophoresed

directly into a composite gel as described in this chapter and elsewhere (10). The gel was

blotted to nitrocellulose and lane 1 was incubated with LF-15 (anti-biglycan antiserum, 1:500)

and lane 2 with LF-30 (anti-decorin antiserum, 1:500). The position of the migration of aggrecan

isolated from bovine cartilage is noted (A).

Gel Electrophoresis and Immunoblotting 153

a lower mobility than the smaller (12). The electrophoresis, therefore, confirmed the

result of the zonal rate electrophoresis, but it failed to explain the nearly threefold

greater Mr of the keratan sulfate-poor species.

The guanidine-HCl extracts of adult bovine tendon subjected to CsCl density-gradient

ultracentrifugation resolved into two populations of proteoglycan, one large and with a

core protein of Mr 200,000 and one small with Mr 48,000. The large proteoglycan subjected

to agarose-acrylamide gel electrophoresis showed three components, which could not be

resolved further (13). Had Western blots for proteoglycans been perfected at that time,

these components might have been more fully characterized.

Canine adult menisci contained two proteoglycans of high Mr that could be resolved

into 2 fractions by agarose/acrylamide gel electrophoresis. These proteoglycans contain

chondroitin sulfate and keratan sulfate, and they bound hyaluronan (14). This study

suggested that there were tissue-specific high-Mr proteoglycans. By contrast to the

proteoglycans of cartilage, which are keratan sulfate rich and keratan sulfate poor, menisci

contain such molecules only in the keratan sulfate-rich form.

In a study of the proteoglycans of the human intervertebral disk, Jahnke and

McDevitt (15) made extensive use of agarose/acrylamide gel electrophoresis. The

hyaluronan aggregating proteoglycans resolved into two well-separated fractions,

which retained their heterogeneity even after hydroxylamine fragmentation and

removal of the hyaluronan-binding domain. The faster migrating species were

resolvable into two components, one of which enriched in keratan sulfate. This

work was extremely labor intensive, using associative and dissociative density-

gradient ultracentrifugation as well as Sepharose 2B and Bio-Gel A-50 m column

chromatography for preparation of samples to be subjected to the electrophoresis.

Much of these labors might have been avoided had these authors used the modifi-

cation of sample preparation (reduction and SDS treatment) introduced in 1985 by

Heinegard et al. (6), which would have allowed instant characterization of the

guanidine•HCl extract.

Agarose/acrylamide gel electrophoresis has also been used in characterizing the

proteoglycans from clinical human specimens (16). In these studies Cs-Szabo et al.

(16) studied the characteristic changes in aggrecan and the small proteoglycans in

cartilage from patients with osteoarthritis and rheumatoid arthritis. Guanidine-HCl

extracts of femoral condyle cartilage were subjected directly to agarose/acrylamide

gel electrophoresis and Western blotting. Samples from patients with rheumatoid

arthritis and some extent also from patients with osteoarthritis lacked certain

aggrecan populations, indicating extensive proteoglycan degradation. The small

proteoglycans biglycan and decorin appeared to be increased by the disease. Repair

processes, particularly in osteoarthritis, were evident through production of fetal-

type aggrecans. This represents an excellent example that takes full advantage of the

powerful technique of agarose/acrylamide gel electrophoresis coupled with Western

blotting.

The ability of articular cartilage to respond to exercise has been well documented,

and this has led many investigators to analyze the proteoglycan content in defined

anatomic sites in different species. One of the more interesting and successful studies,

154 Dodge and Heimer

taking full advantage of agarose/acrylamide gel electrophoresis, was done with

exercised and nonexercised race horses (17). This study, from a technical point of

view, demonstrated that a large number of samples can be analyzed simultaneously.

The complex results, subject to various interpretations, however, showed that

exercised horse had certain loci of articular cartilage that were capable of producing

large polydispersed hyaluronan-binding proteoglycans. In these studies the data

were gleaned from the unique ability of the composite gel to resolve large

proteoglycans and subsequent immunoblotting for a specific domain of aggrecan

(the hyaluronan-binding domain).

Versican, a proteoglycan of high Mr formed in the extracellular matrix, was

surmised to be present through use of agarose/acrylamide gel electrophoresis

and Western blots prepared from the gel. Without an antibody to canine versican,

the authors (18) had to resort to monoclonal antibodies reacting with glycosami-

noglycan epitopes. Chondroitin sulfate was the major glycosaminoglycan

present in a proteoglycan of approximately 500,000 daltons, established by

analysis of the core protein. As the tissue contained hyaluronan and also link

protein, it was postulated that a domain of versican was able to react with

hyaluronan and that the complex might be stabilized by link protein. The actual

identification of canine versican, however, could only be established by partial

amino acid sequencing.

Composite agarose/acrylamide gels have been instrumental in the characterization

and study of various proteoglycans in a number of different cell types and species. The

use of these specialized agarose/acrylamide gels has greatly enhanced our knowledge

of proteoglycan biochemistry, synthesis, and degradation. These gels allow direct

analysis of biological specimens and tissue extracts. Combined with the use of spe-

cific antibodies they potentially and simultaneously can detect distinct proteoglycan

molecules of widely divergent molecular sizes.

2. Materials

1. Sample buffer: 6 M urea, 0.04 M Tris-acetate, pH 6.8, 0.1% Triton X-100, 1% sodium

dodecylsulfate, 5% 2-mercaptoethanol, and 10% glycerol.

2. Agarose (see previous paragraph).

3. Electrode buffer: 0.04 M Tris-acetate, pH 6.8.

4. Triton X-100.

5. TEMED.

6. Large-format vertical protein gel electrophoresis chamber and accessories.

7. Power supply capable of supplying a constant 120 mA.

8. Glass plates with one side frosted (to fit the electrophoresis apparatus).

9. Spacers of 1.5 mm.

10. Transfer apparatus (suitable size to accommodate gel size).

11.Transfer membrane (positively charged Nylon, Immobilon-N [Millipore], or

polyvinylidene fluoride [PVDF] Immobilon-P [Millipore] are recommended; sup-

ported nitrocellulose can also be used).

12. Filter paper (e.g., Whatman no. 3).

Gel Electrophoresis and Immunoblotting 155

3. Method

3.1. Sample Preparation

1. Prepare samples in no more than 10 µL, containing 6 M urea, 0.04 M Tris-acetate, pH 6.8,

0.1% Triton X-100, 1% sodium dodecylsulfate, 5% 2-mercaptoethanol, and 10% glycerol.

Boil samples for 5 min.

3.2. Gel Preparation

1. Before pouring the composite gel solution, a 0.5- to 1.0-cm plug of 10% acrylamide can be

placed at the bottom of the plates after they are prepared. This ensures that the gel will not

slip out during electrophoresis or handling.

2. Prepare the gel mixture in an Erlenmayer flask a final concentration of 0.6% agarose

and 1.2% acrylamide. The agarose solution is prepared with 320 mg of agarose (see

Note 2) and 30 mL of 0.04 M Tris-acetate, 1 mM sodium sulfate, and 1 mM EDTA,

adjusted to pH 6.8.

3. Weigh the flask, add the agarose, and dissolve by heating the flask in a microwave oven.

4. Reweigh and restore to the original weight with distilled water. Keep the flask at 56°C to

prevent gelling.

5. Make the acrylamide solution so that the total concentration of the acrylamide plus

N,N'-methylene-bisacrylamide totals 2.4%, with an acrylamide to bis ratio of 20 to 1.

These two reagents need to be dissolved in 0.04 M Tris-acetate, pH 6.8, containing 2%

Triton X-100 and 3% TEMED.

6. Degas the solution and keep at 56°C.

7. Combine in a small beaker equal volumes of the two solutions, mix, and follow by the rapid

addition of 1% ammonium persulfate.

8. Pour the solution into a preprepared chamber with frosted glass plates separated by 1.5-mm

spacers (see Notes 3 and 4). The chamber should be preincubated at 45°C.

9. The comb is then inserted with care taken to minimize air bubbles and to keep the depth of

the wells at ~0.5 cm.

10. One hour after the casting of the gel, electrode buffer is added to the upper electrode

compartment. The assembly is stored in a cold room or refrigerator.

11. Withdrawing the comb without destroying the wells is the chief hazard of the preparation.

This can be best accomplished by storing the gel in the cold for 24 h and keeping the depth

of the wells to a 0.5-cm maximum. When ready to remove the comb, ease the comb out by

adding a small amount of buffer along the top and pulling straight up.

12. The gel should be prerun for 1 h at 120 mA before loading the samples. When loading the

gel, one well should be reserved for the bromphenol blue tracking dye. The electro-

phoresis should run in a cold room at 120 mA until the bromphenol blue marker has

moved 3 cm. This may take about 3 h (for specifics on dismantling apparatus, see Notes

5 and 6). If not proceeding with immunodection of proteoglycans (transfer described

below), after fixation the gel can be stained directly with toluidine blue. The gel can be

left on one plate and placed in a tray containing fixative (50% methanol, 10% acetic

acid) for 60 min at room temperature followed by a change to a solution of 0.025%

toluidine blue in 3% acetic acid. This requires at least 2 h at room temperature although

it can be protracted to overnight.

13. Destain with 3% acetic acid until the background is suitably clear. The gel can be stored

by transferring to Gelbond film (FMC) as described by the manufacturer (note do not dry

with heat higher than 55°C).

156 Dodge and Heimer

3.3. Western Blotting

1. Transblotting to nitrocellulose is carried out in 25 mM Tris and 192 mM glycine buffer

adjusted to pH 8.3 and containing 5% methanol (reduced methanol improves efficiency of

the transfer of large molecular molecules) (see Note 7). The plates need to be separated

very carefully, usually using bent metal spatulas.

2. The gel adhering to one of the frosted glass plates is then overlaid with a nonsoaked filter

paper and lifted off the plate by peeling backwards.

3. A small amount of transfer buffer should be placed on the top of the gel and the transfer

membrane of choice (prewet) can then be laid on the top (see Note 8). It cannot be moved

once it is placed on the gel, and the air bubbles should be eliminated by gently rolling a pipet

over the membrane covering the gel.

4. A final piece of filter paper (prewet) should be laid on top after any visible bubbles are

eliminated.

5. The transblot should be run with cold buffer in the cold at 75 mA on average for 5–6 h. It

must be kept in mind, however, that a gel may contain both large and small proteoglycans

such as decorin (Mr 1.0 × 10

) and some species of aggrecan (Mr 3.5 × 10

), therefore to

effect a total transfer of each may be nearly impossible.

6. One partial solution to the dilemma is to use two sheets of nitrocellulose and examine

the backup sheet for proteoglycans of lower Mr. Due to the problem of either incom-

plete transfer or loss of proteoglycans, one should attempt quantification of disparate

species with caution.

7. There are no special problems with regard to immunological analysis of the blot and

the remainder of the steps are identical to Western blotting of supports derived from

SDS-PAGE.

8. Additionally, when positively charged Nylon is used, the membrane can be stained with

Alcian blue (0.5% in 3% acetic acid) for 10–20 min followed by rinsing briefly with 5%

acetic acid until the contrast of proteoglycans to background is satisfactory.

4. Notes

1. The technique has largely been underutilized. One reason for this might be that executing all

the steps in the procedure requires practiced hands with some degree of technical patience,

but in reality the method can be run routinely in almost any research laboratory.

2. The agarose to use to achieve the maximum resolving power must be linear (not branched),

with none or low sulfate content and have a low EEO number. Most suppliers (e.g., FMC)

carry a suitable agarose.

3. The cleanliness of the glass plates is imperative to a successful run. Plates should be cleaned

with a concentrated detergent product (e.g., RBS-35 [Pierce]) and rinsed extensively with

O and lastly ethanol.

4. To prevent the gel from slide-out, one or both of the glass plates must be frosted. Addition-

ally, a 0.5- to 1.0-cm plug of ~10% acrylamide will ensure that the gel remains in place

during the electrophoresis and handling.

5. Care should be taken to lift one plate off the gel; generally the nonfrosted plate will lift

off more easily. In the event the gel is sticking, try to loosen it using a gentle stream of

running buffer or water. Plates should be horizontal when trying to separate with the gel

side down. Failure to separate plates without breaking the gel or when there is excessive

sticking is typically due to imperfectly clean plates.

6. Particular care must be taken when handling the composite gel itself when taking the

apparatus apart after the run. Gels composed of agarose and acrylamide are not at all the

Gel Electrophoresis and Immunoblotting 157

consistency of standard Laemli SDS-polyacrylamide gels but are rather crumbly and eas-

ily broken or cracked. The best advice is to pick the gel from the glass plate with a dry

piece of filter paper. Start by overlying the paper and gently peeling it back lifting the gel

from the glass plate.

7. There are no special problems with regard to performing the immunological analysis of

the blotted proteoglycans after the transfer is complete, although care must be taken when

setting up the transfer. Placement of the membrane on the gel must be correct from the

beginning since once placed on the gel it cannot be moved. Additionally, when preparing the

sandwich of gel, membrane, and filter paper, care must be taken not to squeeze the package

or use papers that are too thick, since it is possible to crush and destroy the gel.

8. The use of positively charged Nylon membranes for immunodetection can greatly enhance

the sensitivity of detection (i.e., 10–50 ng of proteoglycan) over that of metachromatic

dyes such as toluidine blue (7). More recently, positively charged Immobilon-N

(Millipore) has been used with nearly equal success.

References

1. Heinegård, D. and Paulsson, M. (1984) Structure and metabolism of proteoglycans, in

Extracellular Matrix Biochemistry (Piez, K. and Reddi, H., eds.), Elsevier, New York,

NY, pp. 277–328.

2. McDevitt, C. A. and Muir, H. (1971) Gel electrophoresis of proteoglycans and

glycosaminoglycans on large-pore composite polyacrylamide-agarose gels. Anal.

Biochem. 44, 612–622.

3. Uriel, J. (1966) Method of electrophoresis in acrylamide-agarose gels. Bull. Soc. Chim.

Biol. 48, 969–982.

4. Peacock, A. C. and Dingman, C. W. (1968) Molecular weight estimation and separation of ribo-

nucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7, 668–674.

5. Sweet, M. B., Thonar, E. J., and Immelman, A. R. (1978) Anatomically determined polydis-

persity of proteoglycans of immature articular cartilage. Arch. Biochem. Biophys. 189, 28–36.

6. Heinegård, D., Sommarin, Y., Hedbom, E., Wieslander, J., and Larsson, B. (1985) Assay

of proteoglycan populations using agarose-polyacrylamide gel electrophoresis. Anal.

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7. Heimer, R. and Sampson, P. M. (1987) Detecting proteoglycans immoblized on positively

charged nylon. Anal. Biochem. 162, 330–336.

8. Heimer, R., Molinaro, L., Jr. and Sampson, P. M. (1987) Detection by

125

I-cationized

cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and

on positively charged nylon 66. Anal. Biochem. 165, 448–455.

9. Heimer, R. (1989) Proteoglycan profiles obtained by electrophoresis and triple

immunoblotting. Anal. Biochem. 180, 211–215.

10. Bashey, R. I., Sampson, P. M., Jimenez, S. A., and Heimer, R. (1993) Glycosaminogly-

cans and chondroitin/dermatan sulfate proteoglycans in the myocardium of a non-human

primate. Matrix 13, 363–371.

11. Roughley, P. J. and White, R. J. (1989) Dermatan sulphate proteoglycans of human articu-

lar cartilage. The properties of dermatan sulphate proteoglycans I and II. Biochem. J. 262,

823–827.

12. Heinegård, D., Wieslander, J., Sheehan, J., Paulsson, M., and Sommarin, Y. (1985) Sepa-

ration and characterization of two populations of aggregating proteoglycans from carti-

lage. Biochem. J. 225, 95–106.

158 Dodge and Heimer

13. Vogel, K. G. and Heinegård, D. (1985) Characterization of proteoglycans from adult

bovine tendon. J. Biol. Chem. 260, 9298–9306.

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high-buoyant-density proteoglycans from semilunar menisci. J. Bone Joint Surg. 68, 55–64.

15. Jahnke, M. R. and McDevitt, C. A. (1988) Proteoglycans of the human intervertebral disc.

Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus.

Biochem. J. 251, 347–356.

16. Cs-Szabo, G., Roughley, P. J., Plaas, A. H., and Glant, T. T. (1995) Large and small proteoglycans

of osteoarthritic and rheumatoid articular cartilage. Arthritis Rheum. 38, 660–668.

17. Palmer, J. L., Bertone, A.L., Malemud, C, J., Carter, B. G., Papay, R. S., and Mansour, J.

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cised and nonexercised horses. Am. J. Vet. Res. 56, 1570–1576.

18. Yamauchi, S., Cheng, H., Neame, P., Caterson, B., and Yamauchi, M. (1997) Identification,

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human tissues as detected by a novel monoclonal antibody against domain III and by in situ

hybridization. J. Histocem. Cytochem. 42, 239–249.

20. Isogai, Z., Shinomura, T., Yamakawa, N., Takeuchi, J., Tsuji, T., Heinegård, D., and Kimata,

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nant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican. Cancer Res. 56,

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body to skeletal keratan sulphate. Importance of antibody valency. Biochem. J. 260, 849–856.

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body that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies

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23. Hughes, C. E., Caterson, B., Fosang, A. J., Roughley, P. J., and Mort, J. S. (1995).

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Alcian Blue Quantitation 159

159

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Quantitation of Proteoglycans in Biological Fluids

Using Alcian Blue

Madeleine Karlsson and Sven Björnsson

1. Introduction

Alcian blue is a tetravalent cation with a hydrophobic core that contains a copper

atom, hence its blue color (1). The four charges allow Alcian Blue to bind to gly-

cosaminoglycans (GAGs) at high ionic strength, in contrast to other cationic dyes,

which are all monovalent. The molecular structure, that is, the plane tetragonal hydro-

phobic core with positive charges attached at its corners, may facilitate formation of

aggregates of several molecules side by side rather than micelle formation. The ionic

strength and presence of other detergents will affect the size of these aggregates in

solution. Such positively charged linear aggregates will bind strongly to some nega-

tively charged polymers such as GAGs, but not to others such as nucleic acids reflect-

ing the conformation of both the polymer and the dye. Alcian blue is not a well defined

substance. There are large differences in solubility and binding characteristics among

different manufacturers but also between batches from the same manufacturer. In addi-

tion, the stain has a limited shelf life and is slowly degraded both in solution and as dry

substance.

Glycosaminoglycans are negatively charged polymers built by repeating units of

disaccharides containing one uronic acid or galactose and one N-acetylglycosamine.

The variation in charge may be very large, since each disaccharide is more or less

sulfated. The ionic bonding between cationic dyes and the negatively charged GAGs

is generally thought to be proportional to the number of negative charges present on

the GAG chain—that is, both sulfate and carboxyl groups. In contrast, studies of the

interaction at different pH and ionic strength (2) revealed that the carboxyl groups are

not involved in the binding of Alcian blue to GAGs. Moreover, the presence of charged

carboxyl groups interferes with binding of Alcian blue to the GAGs. One species of

GAG, keratan sulfate, contains not uronic acid but galactose, and thus lacks the