Iozzo Renato V. Proteoglycan Protocols

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160 Karlsson and Björnsson

carboxyl group. Accordingly, the binding of Alcian blue to keratan sulfate is not pH

dependent, and the amount of dye bound per milligram keratan sulfate is equal to that

of other types of GAGs (2).

The principle is based on the specific interaction between sulfated polymers and

the tetravalent cationic dye Alcian blue at a pH low enough to neutralize all carboxylic

and phosphoric acid groups and at an ionic strength great enough to eliminate ionic

interactions other than those between Alcian blue and sulfated GAGs (2). Hyaluronan,

a nonsulfated GAG, does not react in this assay. There is no interference from proteins

or nucleic acids in this method (see Figs 1 and 9, later, and refs. 2 and 4), in contrast to

the dimethylmethyl blue (DMMB)-method (5,6) or other dye-binding methods. At 0.4

M guanidine-HCl, binding of Alcian blue to GAG chains in proteoglycans is the same

as binding to isolated GAG chains. Thus, the method is equally applicable to

proteoglycans samples as to GAG samples.

A method for simultaneous quantitation and preparation of intact proteoglycans in

biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has

been published (2). The tube/absorbance assay has a measuring range of 1–20 µg of

GAG. Proteoglycans prepared by Alcian blue precipitation are suitable for electrophore-

sis (3) or biochemical analysis. The assay principle can be applied at any scale, from a 50

µL sample in a quantitative assay to a 600-mL sample in large-scale preparation.

Quantitation may also be performed using the dot blot format. In the dot blot/reflec-

tance assay, the Alcian blue–GAG complexes are collected on a polyvinylidene

diflouride (PVDF)-membrane by filtration in a dot-blot apparatus, and the stain is

quantitated as reflectance by scanning densitometry. The dot-blot/reflectance assay

has a measuring range of 10–800 ng of GAG, which is enough to measure the low

contents of proteoglycans in plasma, urine, or wound fluid.

2. Materials

1. Alcian blue, certified for GAG/PG quantitation, Wieslab, Lund, Sweden.

2. Chondroitin-6-sulphate (C-4384) from shark cartilage, keratan sulphate (K3001) from

bovine cornea, heparan sulphate (H7640) from bovine kidney, from Sigma (USA).

3. Capped polypropylene vials (1- or 2-mL), Sarstedt, Sweden.

4. Centrifuge with a fixed-angle rotor for 2-mL vials, and a minimal centrifugal force

12,000g.

5. ELISA photometer equipped with a 605- or 620-nm filter.

6. Syringe (5–10 mL) with a large-bore needle (1.5 mm).

7. MilliBlotD system (#MBBDDD 960) dot-blot apparatus, Biogenex, San Ramon, CA, USA.

8. PVDF membrane (Immobilon-P #IVPH00010), Millipore, USA.

9. A flat-bed scanner, Scanmaker II (Mikrotek Lab, USA) and a PowerMacintosh with

Adobe Photoshop

™

(Adobe Systems, USA) was used for imageing.

2.1. Stock Solutions

1. 10% (v/v) H

: Add 50 mL of H

slowly with stirring to 450 mL of H

O to a final

volume of 500 mL. The solution will be stable for for 1 yr at room temperature.

2. 10% (w/v) Triton X-100 : Dissolve 50g of Triton X-100 in 500 mL of H

O, stir until in

solution. The solution will be stable for for 1 yr at room temperature.

Alcian Blue Quantitation 161

3. Alcian blue stock solution (Alcian blue in 0.1% H

/0.4 M guanidine-HCl ): Add 0.1

mL of 10% H

to 0.5 mL of 8 M guanidine-HCl. Add H

O to 10 mL. Dissolve 0.1-0.2g

(depending on batch) of Alcian blue 8 GS (Wieslab, Sweden). Mix for 2 h to overnight.

Store in the dark at 4°C. The solution will be stable for for 1 mo.

The guanidine-insoluble portion of the stain is removed by centrifugation (12,000g, 15 min)

before each preparation of AB reagent. Dilute 100 µL of the supernatant to 10 mL and

read absorbance at 600 nm. The concentration of Alcian blue in the stock solution should

be adjusted, by addition of 0.4 M guanidine-HCl/0.1% H

, corresponding to an absor-

bance of 1.4 of the diluted stock.

4. 8.0 M guanidine-HCl (Gu-HCl): Dissolve 764 g of guanidine-HCl p.a. (Mw 95.53) in

O and add to 1000 mL. The solution will be stable for for 1 yr at room temperature.

If technical-grade guanidine-HCl is used, it must be stirred overnight with active charcoal

and then filtered. The concentration of a guanidine-HCl may be checked by measuring

the density of the solution: 8.0 M guanidine-HCl has a density of 1.192 g/L.

5. 3.0 M MgCl

: Dissolve 305 g of MgCl

× 6H

O (Mw 203.30) in H

O and add H

O to

500 mL.The solution will be stable for for 1 yr at room temperature.

6. CsC calibrator stock solution (chondroitin-6-sulfate 10mg/mL): Dissolve 20 mg of chon-

droitin-6-sulfate (Sigma C4384) in 2 mL of H

O. The solution will be stable for several

years at –20°C.

7. TG × 10 (0.25 M Tris/1.92 M glycine pH 8.3): Dissolve 12.1 g Tris base and 57.6g of

glycine in a final volume of 400 mL of H

O.The pH should be approximately 8.3, but

need not be adjusted. The solution will be stable for for 1 yr at room temperature.

8. 10% (w/v) SDS: Dissolve 25 g of SDS in a final volume of 250 mL of H

O. The solution

will be stable for for 1 yr at room temperature.

9. 1.0M Tris-acetate, pH 7.4: Dissolve 121 g of Tris base in H

O. Adjust pH to 7.4 with acetic

acid. Add H

O to 1000 mL. The solution will be stable for for 1 yr at room temperature.

2.2. Reagents

1. SAT-reagent (0.3% H

/0.75% Triton X-100): Take 0.3 mL of 10% H

and 0.75 mL

of 10% Triton X-100. Add H

O to 10 mL. The solution will be stable for 1 wk at room

temperature.

2. AB reagent (Alcian blue in 0.1% H

/0.25% Triton): Take 5.0 mL of Alcian blue stock

solution and add 1.0 mL of 10% H

and 2.5 mL of 10% Triton. Add H

O to a final

volume of 100 mL. The solution will be stable for 1 wk at 4°C.

3. DMSO washing solution (40% (v/v) DMSO/0.05M MgCl

): Add 40 mL of DMSO to 40

mL of H

0 and add 1.7 mL of 3.0 M MgCl

. Add H

0 to 100 mL. Store at room tempera-

ture. The solution will be stable for 1 mo.

4. Ethanol washing solution (50% (v/v) ethanol/0.05M MgCl

): Take 52 mL of ethanol

(96%, v/v) and add 1.7 mL of 3.0 M MgCl

. Add H

O to 100 mL. Store at room tempera-

ture. The solution will be stable for 1 mo.

5. Gu-prop-H

O: 4 M guanidine-HCl/33% 1-propanol/0.25% Triton/0.1% Ficoll.) Take 50 mL

of 8.0 M guanidine-HCl, 33 mL of 1-propanol p.a., and 2.5 mL of 10% Triton. Add 0.1 g

of Ficoll. Mix and add H

O to 100 mL. Do not use 2-propanol (isopropanol)! Store at

room temperature. The solution will be stable for 1 yr.

6. Prop-gu: 71% 1-propanol/2.3 M guanidine-HCl): Mix 71 mL of 1-propanol p.a. and 29

mL of 8.0 M guanidine-HCl. Do not use 2-propanol (isopropanol)! Store at room tem-

perature. The solution will be stable for 1 yr.

162 Karlsson and Björnsson

7. Prop-Tris: 75% 1-propanol/0.1 M Tris-Ac. Mix 75 mL of 1-propanol and 10 mL of 1.0 M

Tris/Ac, pH 7.4. Add H

0 to 100 mL. Store at room termperature. The solution will be

stable for 1 yr.

2.3. Sample Preparation

GAGs are stable at 4°C, in the absence of cells. The protein cores of proteoglycans

may be degraded by proteolytic enzymes. The sample must not contain any particles

that sediment during centrifugation or that are insoluble in 0.4 M guanidine-HCl at pH

1.5. Cell debris and insoluble material should be removed by centrifugation (12,000g

for 15 min). If the sample does contain material that precipitates in 0.4 M guanidine-

HCl at pH 1.5, it must be removed by a preceding step (see Subheading 3.4.).

Samples such as plasma or synovial fluid with a high protein and a low proteoglycan

content may be concentrated and purified with an extra preceding Alcian blue precipi-

tation. (see Subheading 3.5.)

3. Methods

3.1. Quantitation, Tube Absorbance Assay (Table 1)

The GAG/PG are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and

collected by centrifugation. Excess stain in the pellet is removed by washing in DMSO.

The GAG/PG–Alcian blue complexes are dissolved and dissociated in a 4 M guani-

dine-HCl/propanol mixture. The amount of GAG/PG is directly proportional to the

Alcian blue concentration measured as absorbance at 605 or 620 nm. The tube/absor-

bance assay has a measuring range of 1–20 µg of GAG. Sample volumes given are

only suggestive. The method can be applied at any scale as long as the final concentra-

tion of guanidine-HCl is 0.4 mol/l at pH 1.5 - 2.0.

1. Put 50 µL of either blank (water), sample, calibrator, or control in 2-mL polypropylene

centrifuge tubes.

2. Add 50 µL of 8 M Gu-HCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl are

analyzed without further addition of guanidine-HCl. Take 100 µL of the sample and go

directly to step 3.

3. Add 50 µL of SAT reagent. Mix and leave for 15 min.

4. Add 750 µL of cold AB reagent. Mix and leave for 15 min to overnight at 4°C.

5. Centrifuge for 15min at 12,000g. Remove supernatant and discard. Care should be taken

that pellets are left intact when supernatants are removed by suction. The use of a manu-

Table 1

Preparation of Calibrators, Tube Absorbance Assay

Calibrator (mg/mL) Dilution H2O (µL) µg GAG in 50µL

0.4 80µl CsC stock 1920 20 µg

0.2 1000µl 0.4mg/mL 1000µl 10 µg

0.1 1000µl 0.2mg/mL 1000µl5 µg

0.05 1000µl 0.1mg/mL 1000µl 2.5 µg

0.025 1000µl 0.05mg/mL 1000µl 1.25 µg

0.0125 1000µl 0.025mg/mL 1000µl 0.0625 µg

Alcian Blue Quantitation 163

ally operated syringe for suction and immediate removal of the supernatants after cen-

trifugation is therefore recommended. The tube should be held at an angle and in such a

way that the pellet and needle are visible during the entire operation. The needle should be

gradually lowered with the meniscus during suction and never be allowed to touch the pellet.

6. Add 500 µL of DMSO solution to the pellet. Mix thoroughly until the pellet is suspended

in the washing solution. Mix for 15 min on a shaker.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 500 µL Gu-prop-H

O solution to the pellet. Mix for 15 min on a shaker. Check that

the pellet is completely dissolved.

9. Dispense 2 × 240 µL of the supernatant into an ELISA-microplate. Read absorbance at

605 nm.

10. Plot the absorbances against amount of GAG in each calibrator (0–20 µg). The plot should be

a perfectly straight line with an absorbance of approximately 2.0 at 20 µg and blank values

below 0.05 (Fig. 1). High blank values may be caused by insoluble, degraded stain. The dry

Alcian blue stain has a limited shelf life, and some batches are not suitable for biochemical

work. The stock solution also has a limited shelf life, and it is important that the AB stock

solution be centrifuged in order to remove the insoluble portion of Alcian blue. Fit a linear

equation and calculate the amount of GAG in each sample. Alternatively, if only a single

calibrator concentration is used, a factor is calculated by dividing the calibrator (µg) by the

corresponding absorbance. The absorbance of the unknown samples is then multiplied by

the factor to obtain micrograms of GAG in each sample.

All commercial sulfated GAG samples (CsA, CsC, Ds, Ks , Hs) should give a similar colour

yield. HA, Hyaluronan, DNA, and RNA do not react with Alcian blue at all. There is no

interference from proteins in blood plasma (Fig. 1).

3.2. Preparation for Electrophoresis Without Removal of Alcian Blue

The GAG/PGs are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and

collected by centrifugation. Excess stain in the pellet is removed by washing in DMSO.

Fig 1. Tube assay of different GAG in blood plasma. Commercial preparations of glycosami-

noglycans were dissolved in water or in blood plasma (0–0.4mg/mL) and was quantitated with

Alcian Blue using the tube assay. -❏- Chondroitin-6-sulphate; -䉫- Keratan sulphate; -❍-

Heparan sulphate; Open symbols: GAG dissolved in water. Closed symbols: GAG dissolved in

blood plasma.

164 Karlsson and Björnsson

The GAG/PG/Alcian blue complexes are dissolved directly in an SDS buffer and ana-

lyzed by electrophoresis.

The SDS used to dissolve the GAG/PG–Alcian blue complexes will move in front

of any GAG/PG, Fig. 2. The relative mobility of the SDS–Alcian blue complexes will

depend on the ratio of SDS bound per Alcian blue molecule.

This method is useful for revealing losses during the propanol precipitation step (see step

9 and 10 under Subheading 3.3.). Any losses can be detected by comparing the electrophore-

sis patterns of the same samples obtained as described under Subheading 3.2. and 3.3.

1. Take 100 µL of either blank (water), sample, calibrator or control to 2 mL polypropylene

centrifuge tubes.

2. Add 100 µL of 8M GuHCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl are

analyzed without further addition of guanidine-HCl. Take 200 µL of the sample and go

directly to step 3.

3. Add 100 µL of SAT reagent. Mix and leave for 15 min.

4. Add 1500 µL of cold AB reagent. Mix and leave for 15min - overnight at 4°C.

5. Centrifuge for 15min at 12000g. Remove supernatant and discard.

6. Add 750 µL of DMSO solution to the pellet. Mix thoroughly; the pellets should be sus-

pended in the washing solution. Mix for 15 min.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 50 µL of TG × 3/2.5% SDS to pellets (sample, calibrator and control).

9. Quantitation may be performed as follows. Put 5 µL of each dissolved pellet on a

microtiter plate. Add 100 µL TG × 3/0.5% SDS to each well and mix. Use reverse

pipetting technique to minimize formation of bubbles. Read absorbance at 600 nm.

10. The amount of sample to be applied to each lane is calculated as follows. Divide the

calibrator (µg) by the corresponding absorbance. The absorbance of the unknown sample

is then multiplied by the factor to obtain micrograms of GAG in each sample. About 3–10

µg are suitable for electrophoresis. Add TG × 3/0.5% SDS to a final volume of 50 µL.

Fig. 2. Alcian blue precipitation without stain removal. Proteoglycans in 4 M Gu-HCL

extracts of cartilage and skin were precipitated with by Alcian blue as described under Sub-

heading 3.2. The samples were analyzed by electrophoresis in 1% agarose and stained with AB

reagent containing 0.4 M GuHCl. Lane 1; chondroitin-6-sulfate. Lane 2; 4 M Gu-HCl extract of

bovine nasal cartilage; Lane 3; 4 M Gu-HCl extract of human skin. The position of AB–SDS

complexes is indicated in the figure.

Alcian Blue Quantitation 165

11. Add 25 µL of liquified 1% agarose with 15% glycerol. Heat at 105°C for 5 min and apply

75 µL of the hot sample on each lane on a 1% agarose gel in 0.1 M Tris-Ac. Electro-

phorese for 1 h at 100 V, until the BFB front has moved 60 mm. Stain in AB-reagent with

0.4 M GuHCl (see Fig. 2.).

3.3. Preparation with Removal of Alcian Blue

The GAG/PGs are allowed to precipitate in 0.4 M guanidine-HCl at pH 1.5 and col-

lected by centrifugation. Excess stain in the pellet is removed by washing in DMSO. The

GAG/PG–Alcian Blue complexes are dissociated and dissolved in a 4 M guanidine-HCl/

propanol mixture. The GAG/PGs are precipitated by increasing the propanol concentra-

tion, with addition of a 71% propanol/guanidine-HCl mixture, and are then recovered by

centrifugation. The amount of GAG/PG is directly proportional to the Alcian blue con-

centration in the supernatant, measured as absorbance at 605 nm.

1. Put 100 µL of either blank (water), sample, calibrator, or control in 2-mL polypropylene

centrifuge tubes.

2. Add 100 µL of 8 M Gu-HCl. Mix and leave for 15 min. Samples in 4 M guanidine-HCl

are analyzed without further addition of guanidine-HCl. Take 200 µL of the sample and

go directly to step 3.

3. Add 100 µL of SAT reagent. Mix and leave for 15 min.

4. Add 1500 µL of cold AB reagent. Mix and leave for 15min to overnight at 4°C.

5. Centrifuge for 15 min at 12000g. Remove supernatant and discard.

6. Add 750 µL of DMSO solution to the pellet. Mix thoroughly; the pellets should be sus-

pended in the washing solution. Mix for 15 min.

7. Centrifuge as in step 5. Remove supernatant and discard.

8. Add 250 µL of Gu-prop-H

O to the pellet. Mix for 15 min or until the pellet is dissolved.

9. Add 1750 µL of Prop-gu. Mix and leave for 60 min . Loss of GAG/PG may occur during

propanol precipitation/centrifugation. Most proteoglycans will precipitate quantitatively

at a rather low propanol concentration but isolated GAG chains may not precipitate quan-

titatively. Thus, keratan sulfate require higher propanol concentrations than other GAGs,

Fig. 3. Any losses can be detected by comparing the electrophoresis patterns of the same

samples without propanol precipitation (see Subheading 3.2.) and with propanol precipi-

tation (see Subheading 3.3.).

10. Centrifuge for 15 min at 12000g. Remove the supernatant using a syringe. Read absor-

bance at 605 nm of the supernatant. The expected absorbance of the supernatant is 1.0 at

0.4 mg/mL (40 µg).

11. Add 750 µL of Prop-Tris to the pellet. Mix.

12. Centrifuge for 5 min at 12,000g. Remove the supernatant and discard. The pellet will

always contain some insoluble degraded Alcian blue. The dye in the pellet is not water

soluble, and can be sedimented by centrifugation. The concentration of propanol in the

Prop-gu solution, used for precipitation of GAG/PG, will affect the amount of Alcian

blue remaining in the pellet, Fig. 4. The amount of Alcian blue in the pellet also depends

on the GAG and is especially high with keratan sulfate, Fig. 4.

13. Dissolve the pellet in 50 µL TG × 3/0.1% SDS. The pellet may also be dissolved in water

or a buffer without SDS.

14. The amount of proteoglycan needed for electrophoresis may be calculated as above (see

Subheading 3.2., step10) and analyzed by electrophoresis (see Fig. 5).

166 Karlsson and Björnsson

Fig. 3. Removal of Alcian blue stain: precipitation of GAG at different concentrations of

propanol. GAGs were precipitated by Alcian blue and the precipitates were dissolved in 250

µL of a 4 M GuHCl/33% propanol mixture as described under Subheading 3.3. GAGs were

precipitated by adding 1750 µL of a propanol/GuHCl mixture. The following propanol/GuHCl

mixtures were used: 67% propanol 2.66 M GuHCl; 69% propanol/2.48 M GuHCl; 71% pro-

panol/2.32 M GuHCl; 73% propanol/2.16 M GuHCl; 75% propanol/2.0 M GuHCl. The precipi-

tated GAGs, recovered by centrifugation, were again quantitated using the method of

Subsection 3.1. and the recovery calculated. -❏- Chondroitin-6-sulfate; -䉫- Keratan sulfate; -

❍- Heparan sulfate.

Fig. 4. Removal of Alcian blue stain: coprecipitation of Alcian blue with GAG at differ-

ent concentrations of propanol. Same experiment as in Fig. 3. The amount of Alcian blue

that co-precipitated with GAGs after centrifugation was quantitated as absorbance at 620 nm

of the solubilized pellet. -❏- Chondroitin-6-sulfate; -䉫- Keratan sulfate; -❍- Heparan sulfate.

Alcian Blue Quantitation 167

3.4. Purification of Difficult Samples

Some samples may contain material that precipitates at a low pH or that binds

unspecifically to Alcian blue. Such material may be removed by an extra Alcian blue

precipitation step as described below.

1. Put 100 µL of either blank (water), sample, calibrator, or control into 2-mL polypropy-

lene centrifuge tubes.

2. Add 100 µL of 8 M Gu-HCl. Mix and leave for 15 min.

3. Add 100 µL of SAT reagent. Mix and leave for 15 min.

4. Add 1500 µL of cold AB reagent. Mix and leave for 15 min to overnight at 4°C.

5. Centrifuge for 15 min at 12000g. Remove supernatant and discard.

6. Dissolve the pellet in 100 µL of H

O + 100 µL of 8 M GuHCl + 100 µL of SAT reagent.

Mix for 15 min.

7. Centrifuge for 15 min at 12000g. Transfer the supernatant to a new vial. Discard the

pellet. Alternatively, the pellet may be washed in DMSO-washing solution and dissolved

in TG × 3/2.5% SDS and analyzed by electrophoresis to verify that no GAG/PG are

present in the pellet, Fig. 6.

8. Continue at step 4 under Subheading 3.2. or 3.3.

3.5. Large-Scale Preparation

Large-scale preparation consists of two successive precipitations with Alcian blue

and is suitable for dilute samples such as plasma and urine. In the first precipitation step

the GAG/PGs are concentrated. When the first precipitate is dissolved and centrifuged,

any acid-insoluble material is left behind in the pellet. The supernatant will contain the

proteoglycans, which are further purified by a second Alcian blue precipitation.

The GAG/PG–Alcian blue complexes recovered after the second precipitation are

dissolved and dissociated in a 4 M guanidine-HCl/propanol mixture. The GAG/PG is

recovered free from stain by selective propanol precipitation.

Fig. 5. Alcian blue precipitation with stain removal. Proteoglycans in four different human

synovial fluids were precipitated by Alcian blue as described under Subheading 3.3. The

samples were analyzed by electrophoresis in 1% agarose and stained with AB-reagent contain-

ing 0.4 M Gu-HCl.

168 Karlsson and Björnsson

Table 2

Sample of Results with Large Scale Preparation Method

Dispensed Total Total Approx.

volume volume absorbance amount of

Fraction Absorbance (mL) (mL) GAG/PG µg

Step 5—supernatant 1.86 0.1 3.75 69.7 348

Step 8—supernatant 0.51 0.2 10 25.5 127

Step 11—supernatant 1.97 0.24 6.8 55.8 279

Final quantitation of 100 µL1.75 × 5 0.24 0.5 45.5 227

The amount of GAG is directly proportional to the Alcian blue concentration in the

supernatant, measured as absorbance at 605 or 620 nm.

Recovery of GAG/PG throughout the procedure is approximately 70%. The main

losses are in the propanol precipitation/centrifugation step (see Table 2). The size

distribution and purity of the purified preteoglycans could be analyzed by agarose

electrophoresis and parallel lanes stained with Coomassie Brilliant blue and Alcian

blue, Fig. 7. The proportions given below can be applied at any scale.

1. Put a 25 mL sample in a 50 mL propylene tube. Add 2.5 mL 8 M Gu-HCl and 1.25 mL

10% Triton. Mix and leave for 15 min.

2. Add 2.5 mL of the Alcian blue stock solution. Mix carefully and leave for 15 min.

3. Add 0.67% (v/v) H

up to 50 mL. Leave overnight at 4°C.

4. Centrifuge at 3300g, 60 min. Remove the supernatant and discard. Let the tube drain

upside down.

Fig 6. Purification of difficult samples. Proteoglycans in human blood plasma were precipi-

tated with Alcian blue and the pellets extracted with a mixture of 4 M Gu-HCl/SAT-reagent

and centrifuged as described under Subheading 3.4. The extracted proteoglycans in the super-

natant were reprecipitated as described under Subheading 3.2. The pellet was washed in DMSO

washing solution and dissolved in TG × 3-2.5% SDS. The samples were analyzed by electro-

phoresis in 1% agarose and stained with AB-reagent containing 0.4 M GuHCl. Lane 1; superna-

tant from Subheading 3.4., step 7; Lane 2; pellet from Subheading 3.4., step 7. The position

of AB - SDS complexes is indicated in the figure.

Alcian Blue Quantitation 169

Fig. 7. Large scale preparation. Proteoglycans were prepared from human blood platelets

with two successive precipitations with Alcian Blue as described under Subheading 3.5. The

purified proteoglycans were analyzed by electrophoresis in 1% agarose.Lane A: stained with

AB - reagent with 0,4 M GuHCl. Lane B: stained with Coomassie Brilliant Blue in 5% (v/v)

acetic acid - 25% (v/v) ethanol.

Fig. 8. Dot-blot assay. Heparan sulfate calibrator (0–80 mg/L), dissolved in water (A) or in blood

plasma (B), was precipitated with Alcian blue and the GAG precipitates collected on a PVDF mem-

brane by filtration in a dot-blot apparatus. The membrane was scanned on a single layer of clear plastic.