Iozzo Renato V. Proteoglycan Protocols

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Purification from Mineralized Tissues 23

3. Profile aliquots from fractions with significant A 280-nm absorbance by SDS PAGE (see

below). Profile proteoglycan elution positions by a modified carbazole (uronic acid) assay

(see Note 7).

3.4. Size-Exclusion Chromatography

1. Molecular size separations are performed using two different systems. The columns and

flow rates used are a Superose 6 HR 10/30 FPLC column at a flow rate of 0.25 mL/min and

a Phenomenex BioSep SEC S-4000 column at a flow rate of 0.5 mL/min. The Superose 6

column is used to further isolate individual proteoglycans, while the BioSep SEC S-4000

column is used for final purification and estimation of size.

2. Pooled fractions from anion exchange are concentrated by PEG precipitation (see above).

3. Pellets are resuspended in the formamide equilibration buffer and injected onto the Superose

6 column.

4. 1-min fractions are collected as absorbance at 280 nm is monitored and 50-µL aliquots of

each fraction are diluted to 250 µL with H

O and taken for the carbazole assay (see below)

to profile uronic acid-containing proteoglycans.

5. Pooled fractions that have been subjected to anion exchange and subsequent size-exclusion

chromatography on Superose 6 are concentrated by PEG precipitation.

6. The pellet is resuspended in the 6 M guanidine-containing buffer and resolved on the

BioSep SEC S-4000 column. Again, absorbance is followed at 280 nm and aliquots are

analyzed for uronic acid (proteoglycan) content.

7. Both columns are calibrated using protein standards (8).

3.5. Electrophoretic Analysis

3.5.1. Pouring Gradient Gels (see Note 8).

1. 3–15% gradient large gels (16 × 14 × 0.15 cm)

a. 14.6 mL 3% in left chamber.

b. 14.5 mL 15% in right chamber with mixing flea stir bar.

c. Add in order: 35 µL 10% APS to both chambers (mixing with pipet in chamber

without stir bar); 2.75 µL TEMED to 3% side; 2.5 µL TEMED to 15% side.

d. Open stopcocks and start pumps (flow rate

< 1 mL/min).

e. It should take about 40–45 min to pour the separating gel and another 40 min to

polymerize.

f. Top off gel with saturated butanol until ready to pour stacking gel.

2. 3% stacking gel for large gels (16 × 14 × 0.15 cm)

a. To 20 mL of “special 3% stacking gel solution” add: 300 µL APS; and 30 µL TEMED.

3. Small gels (10 × 10.5 × 0.15 cm)

a For small gels, 5 mL of working gel solution is used per chamber of the gradient

former.

b. Each chamber receives 25 µL APS.

c. The 3 % acrylamide working solution receives 2.6 µL TEMED, while the 15%

working solution receives 2.5 µL of TEMED.

d. The 5 mL of stacking gel working solution (special 3%) receives 25 µL APS and

5 µl TEMED.

3.5.2. Running Gradient Gels

1. Samples are reduced with 2 mM dithiothreitol and heated at 100

C for 10 min before

loading. Samples in the formamide buffer do not require the addition of gel sample

buffer and can be loaded directly onto the gel.

24 Fedarko

2. Electrophoresis is carried out on SDS slab gels (16 ×14 × 0.15 c m) at a constant

current of 9 mA per gel for 14 h (30 mA/gel in 6 h with water a buffer cooling system)

or until the prestained carbonic anhydrase molecular weight marker (30,000 daltons)

reaches the bottom of the gel.

3. For small gels (10 ×10.5 × 0.15 cm), electrophoresis is carried out at 25 mA/gel in 2 h.

4. At the end of electrophoresis, gels are rinsed in the 25% isopropanol through 4 changes

over a 12-h period to remove SDS. Residual SDS will interfere with Stains-All solubility.

5. Gels are reacted with the Stains-All working solution in a light-protected container.

6. Once bands are readily apparent, the gels are destained in H

O. Stains-All reacts with

proteins and proteoglycans to generate different colored bands based on band compo-

nent acidity. An advantage of Stains-All over other proteoglycan–glycosaminoglycan

staining procedures is that it also enables contaminating proteins and glycoproteins to be

visualized.

3.6. Modified Carbazole Assay

1. Cool samples and standards (250 µL) in an ice bath.

2. Add 1.5 mL of ice-cold sulfuric acid-sodium tetraborate reagent with mixing and cooling

on ice.

3. Heat the mixtures at 100ºC for 10 min.

4. Cool rapidly in the ice bath.

5. Add 50 µL of carbazole reagent and mix well.

6. Reheat at 100

C for 15 min.

7. Cool rapidly to room temperature and determine the absorbance at 525 nm.

The combination of anion-exchange chromatography and size-exclusion

chromatography enables separation of the three major proteoglycans present in

bone—a versican-like proteoglycan, biglycan, and decorin. The small leucine-rich

proteoglycans, biglycan and decorin, elute early on the salt gradient in anion

exchange and are included and elute later on the size-exclusion columns.

4. Notes

1. Fresh formamide should be used and, once opened, the bottle stored tightly capped at –20ºC

until further use.

2. Omnifit glass columns were adapted for HPLC with Tefzel tubing (1/16-in. outer diam-

eter × 0.01 in. internal diameter) by boring out the ferrules with a 1/16-in drill bit (6).

3. Stains-All, or 1-ethyl-2-|3-(1-ethylnaphtho|1,2d|-thiazolin-2-ylidene)-2-, ethylpro-

penyl|naphtho|1, 2d|thiazolium bromide, is available from Eastman Kodak Co. with a

catalog number of 2718. An alternative to Stains-All profiling of proteoglycans is

Alcian blue staining (9).

4. Working solutions are stable for 3–4 mo when stored at 4ºC in flasks wrapped with aluminum

foil (to eliminate light exposure).

5. Following incubation with demineralizing buffer, it is common in bone samples derived

from adult animals to have a significant amount of insoluble material that pellets after

centrifugation. This majority of this material consists of highly cross-linked type I col-

lagen, though minor amounts of matrix glycoproteins and proteoglycans may also be

present. In general, the older the sample donor, the higher are the levels of insoluble

cross-linked material.

Purification from Mineralized Tissues 25

6. Incubation of the 40% PEG in water suspension at 50ºC facilitates dissolution.

7. Alternatives to the carbazole assay to follow the uronic acid content as a marker of the

glycosaminoglycans in proteoglycans would be one of the numerous stoichiometric dye

binding assays such as Safranin O (10) or 1,9-dimethylmethylene blue (11).

References

1. Termine, J. D., Belcourt, A. B., Miyamoto, M. S., and Conn, K. M. (1980) Properties of

dissociatively extracted fetal tooth matrix proteins. II. Separation and purification of fetal

bovine dentin phosphoprotein. J. Biol. Chem. 255, 9769–9772.

2. Termine, J. D., Belcourt, A. B., Conn, K. M., and Kleinman, H. K. (1981) Mineral and

collagen-binding proteins of fetal calf bone. J. Biol. Chem. 256, 10,403–10,408.

3. Fisher, L. W., Termine, J. D., Dejter, S. W., Jr., Whitson, S. W., Yanagishita, M., Kimura,

J. H.; Hascall, V. C.; Kleinman, H. K.; Hassell, J. R.; and Nilsson, B. (1983) Proteoglycans

of developing bone. J. Biol. Chem. 258, 6588–6594.

4. Fisher, L. W., Hawkins, G. R., Tuross, N., and Termine, J. D. (1987) Purification and partial

characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin

from the mineral compartment of developing human bone. J. Biol. Chem. 262, 9702–9708.

5. Beresford, J. N., Fedarko, N. S., Fisher, L. W., Midura, R. J., Yanagishita, M., Termine, J.

D. and Robey, P. G. (1987) Analysis of the proteoglycans synthesized by human bone cells

in vitro. J. Biol. Chem. 262, 17,164–17,172.

6. Fedarko, N. S., Termine, J. D., and Robey, P. G. (1990) High-performance liquid chromato-

graphic separation of hyaluronan and four proteoglycans produced by human bone cell

cultures. Anal. Biochem. 188, 398–407.

7. Mayes, E. L.V. (1984) Protein, in Methods in Molecular Biology (Walker, J. M., ed.),

Humana, Totowa NJ, pp. 5–12.

8. Chaplin, M. F. (1986) Monosaccharides, in Carbohydrate Analysis: A Practical Approach.

(Chaplin M.F. and Kennedy, J. F. eds.), IRL Press, Oxford, pp. 1–36.

9. Wall, R. S.and Gyi, T. J. (1988) Alcian blue staining of proteoglycans in polyacrylamide gels

using the “critical electrolyte concentration” approach. Anal. Biochem. 175, 298–299.

10. Carrino, D. A., Arias, J. L., and Caplan, A. I. (1991) A spectrophotometric modification

of a sensitive densitometric Safranin O assay for glycosaminoglycans. Biochem. Int. 24,

485–495.

11. Muller, G. and Hanschke, M. (1996) Quantitative and qualitative analyses of proteoglycans

in cartilage extracts by precipitation with 1,9-dimethylmethylene blue. Connect. Tissue

Res. 33, 243–248.

Perlecan from Endothelial Cells 27

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Purification of Perlecan from Endothelial Cells

John Whitelock

1. Introduction

Perlecan, the major heparan sulfate (HS) proteoglycan of basement membranes and

other connective tissues, is a modular molecule with five structural domains. It has

been isolated from various sources including kidney and placenta, but most com-

monly from the mouse Englebroth Holm Swarm (EHS) tumor (1,2). It has also been

purified from cultured cell lines such as endothelial cells (3) or fibroblasts (4). When it

has been extracted from tissue or from the extracellular matrix of cultured cells, it has

been first solubilized using chaotropic agents such as urea or guanidine. The effect of

these dissociating agents on the structure and function of isolated proteoglycans is

not known, but it is thought that some of the denaturation may be irreversible. Human

perlecan is found predominantly as a heparan sulfate proteoglycan, but has been

isolated from carcinoma cell lines as an undecorated protein core and from placental

tissue decorated with chondroitin sulfate/dermatan sulfate. The molecular weight of

the protein core is approximately 470 kDa, while the size of the mouse homolog is

around 400 kDa. Domain I is the N-terminal domain, and it contains a cluster of three

potential glycosaminoglycan attachment sites. Domain II has homology to the LDL

receptor, with strict conservation of the cysteine residue positions. Domain III shares

homology with the short arm of the laminin α-chain. Domain IV is the largest domain

with a molecular weight in excess of 200 kDa and is made up of numerous immunoglo-

bulin-like repeats similar to those found in the neural cell adhesion molecule. Domain

V is the C-terminal domain, and is made up of three regions with homology to the

globular domains of the laminin α-chain, interspersed with four epidermal growth

factor-like repeats.

28 Whitelock

2. Materials

1. Human endothelial cells were isolated from either umbilical veins or arteries as described

previously (5). Dr. J. Gamble provided C11-STH, the endothelial cell line at the Hanson

Cancer Center in Adelaide, Australia.

2. Endothelial cell growth medium: For 1 L of Medium 199 (Earles salts), open 1 packet of

Medium 199 powder (Gibco) and dissolve powder in 900 mL of pyrogen-free H

(Baxter), add 2.2 g of NaHCO3, 100 U of penicillin, and 100 µg of streptomycin. Adjust

the pH to 7.2, make the total volume up to 1 L, sterilize by filtration using a 0.22-µm filter

and store at 4°C. Before using the medium in cell cultures, add 0.3g/L of bovine brain

extract (BBE), prepared as described (6) (see Note 1), 1 mg/L of heparin (Sigma cat. no.

H3149) (see Note 2), 15% v/v fetal bovine serum (see Note 3).

3. Flasks that were used to grow endothelial cells were first coated with 10 µg/mL of

fibronectin for 2 h at 37°C. A 2% gelatin solution can be used in place of fibronectin.

4. DEAE Sepharose column (Pharmacia).

5. Anti-perlecan immunoaffinity column. Weigh out 2 g of dry CnBr-activated Sepharose 4B

matrix (Pharmacia). This makes a column of bed volume 7 mL. Swell and wash the powder

in 1 mM HCl and then equilibrate the gel with approx. 50 mL of 0.1 M NaHCO

, 0.5 M

NaCl, pH 8.3 (coupling buffer). Dialyze the anti-perlecan monoclonal antibody (A71, A74,

or A76) against the coupling buffer (allow approx. 5 mg of antibody per milliliter of swelled

Sepharose). Mix the antibody solution with the prepared gel and mix overnight at 4°C on a

rocking platform. The ratio of buffer to swelled gel should be at least 2/1 to achieve efficient

mixing. This time can be shortened to 4 h at room temperature. Centrifuge at 1000g,

aspirate the supernatant, and wash the gel on a sintered glass funnel under vacuum. Block

the remaining active binding sites by incubating the gel with 1 M ethanolamine, pH 8.0

(this should be made fresh) for 2 h at room temperature on a rocker. Centrifuge at 1000g,

aspirate the supernatant, and wash the gel with coupling buffer. Perform alternate washes

of the elution buffer to be used and the coupling buffer. Equilibrate the column in phos-

phate-buffered saline and store the column at 4°C

6. Dialysis tubing—10,000-Mr cutoff membrane (Spectropor).

7. Sucrose.

8. Superose

6 prepackaged HR 10/30 column and Pharmacia FPLC

system.

9. Coomassie Plus Protein Assay Kit (Pierce).

10. SDS-PAGE gradient gels, reagents, and electrophoretic equipment (Bio-Rad).

11. Composite agarose-acrylamide gels (see Note 4).

12. Anti-perlecan protein core antibodies; A71 (specific for domain I), A74 (specific for

domain V) and A76 (not characterized) (7).

13. Anti-heparan sulfate antibody, 10E4 (Seikagaku).

14. Anti-chondroitin sulfate antibody, CS-56 (Sigma).

15. ELISA plates, reagents, and microtiterplate reader

3. Methods

3.1. Human Endothelial Cell Culture

1. Cultures of human arterial and venous cells are maintained by replacing the conditioned

medium three times every week.

2. The cells can also be grown in roller culture bottles (900 cm

). These will need to be coated

with fibronectin (10 µg/mL; 10 mL per roller bottle) for 2 h at 37°C before seeding the cells.

3. Roller cultures are fed twice weekly with 120 mL of endothelial cell growth medium.

Perlecan from Endothelial Cells 29

4. Once the cells condition the medium, it is removed, centrifuged at 1500g for 10 min to

remove cell debris and stored at –20°C. (see Notes 5 and 6).

3.2. DEAE Chromatography

1. Equilibrate a 100 mL bed volume DEAE Sepharose column with approximately 4 vol-

umes of 20 mM Tris, 250 mM NaCl, 10 mM EDTA, 1 mM benzamidine, pH 7.5 (DEAE

running buffer) at 4°C.

2. Thaw and filter the conditioned medium (approx. 2-L batches) using either glass wool or

a sintered funnel over a vacuum.

3. Apply the medium to the column at a flow rate of 1–2 mL/min.

4. Wash the medium through the column with the DEAE running buffer and continue to wash

the column with this buffer until a baseline is achieved (measuring the absorbency at a

wavelength of 280 nm) (see Note 7).

5. Elute bound molecules (including the perlecan) from the column with 20 mM Tris, 1 M

NaCl, 10 mM EDTA, 1 mM benzamidine, pH 7.5 into a collection vessel which is on ice.

6. Pool the perlecan containing fractions (approx. 60–80 mL) and store at 4°C.

7. Remove a small sample (~200 µL) for testing in the ELISA based screening assay.

8. Regenerate the DEAE column by washing it with 2 volumes of 20 mM Tris, 2 M NaCl,

10 mM EDTA, 1 mM benzamidine, pH 7.5 and reequilibrating it with the DEAE run-

ning buffer.

3.3. Anti-Perlecan Immunoaffinity Chromatography

1. Equilibrate the column with 20 mM phosphate buffer with 1 M NaCl, pH 7.5 (immuno-

affinity running buffer).

2. Apply the pooled perlecan-containing fractions directly to the immunoaffinity column and

recirculate over the column for approx. 6 h or overnight at 4°C, at a flow rate of 1 mL/min.

3. Keep the flow through for testing in the ELISA-based screening assay.

4. Wash the column with approx. 50 mL of the immuno-affinity running buffer or until a

baseline is achieved.

5. Elute the bound perlecan from the immunoaffinity column with either 0.1 M Tris, pH 7.5

containing 3 M MgCl

or PBS containing 6 M urea. (see Note 8).

6. Pool the perlecan peak (approx. 15 mL).

7. Wash the column with the immunoaffinity running buffer. (Another perlecan peak may

elute at this stage; the two peaks should be pooled.)

8. The column is washed with 50 volumes of immunoaffinity running buffer containing 0.08%

NaN

and stored at 4°C.

9. Dialyze the perlecan containing peak against three changes of PBS at 4°C overnight and

then concentrate to approx. 1–2 mL by removing the PBS through the dialysis tubing by

laying it on a bed of sucrose.

10. Once the volume is reduced to approx. 1–2 mL, the clips on the dialysis tubing can be

readjusted (to prevent reswelling) and redialyzed against PBS overnight at 4°C to remove

any sucrose.

11. The immuno-purified perlecan can be assayed for purity and quantity, and stored in small

aliquots at –70°C in siliconized tubes (see Note 9).

3.4. Gel Filtration Chromatography

1. Equilibrate a Superose 6 prepackaged HR 10/30 (Pharmacia) column with 0.5 M

COONa, 0.05% Tween-20, pH 7.5 at 0.4 mL/min.

30 Whitelock

2. Set the fraction collector to collect fractions every 2 min (0.8-mL fraction volume).

3. Apply sample via the loop while maintaining the flow rate at 0.4 mL/min.

4. Fractions can be monitored for either the presence of proteins by measuring the absor-

bency at a wavelength of 280 nm, or for radioactivity by taking a sample and mixing it

with scintillant and counting.

5. This method is essentially as described by Melrose and Ghosh (8). It is particularly suit-

able for separating biosynthetically labeled perlecan from free label (see Fig. 1), as well

as separating

H-labeled or

-labeled HS chains that are liberated from the protein

core of perlecan by alkaline borohydride elimination (see Fig. 2).

6. Perlecan-enriched fractions can be prepared using this methodology. This is particularly

suitable if immuno-affinity chromatography is not possible. It is important to remember that

these fractions will also contain other large molecules such as the chondroitin sulfate

proteoglycan, versican. This can be monitored by the screening of fractions with the anti-

chondroitin sulfate monoclonal antibody, CS-56 (see Note 10).

3.5. Preparation of Perlecan HS (Alkaline Borohydride Elimination)

1. Prepare 2 M NaBH

2. Prepare 0.1 M KOH.

3. Mix an equal volume of the above solutions and add this to a volume of labeled perlecan

solution in the ratio 2/1 v/v (NaBH

/KOH:perlecan).

4. Cover with parafilm, and incubate overnight at 45°C.

5. Neutralize with an equal volume of 3.6 M CH

COOH (see Note 11).

Fig. 1. Elution profile of immunopurifed human perlecan using a Superose 6 10/30

prepacked column. Samples of

H-labeled perlecan were run through a Superose 6 10/30 col-

umn as described. HUAEC-derived perlecan is shown by the dashed line, whereas HUVEC-

derived perlecan is shown by the unbroken line. The V

of the column was 8 mL (fraction 10),

whereas the V

was 20 mL (fraction 25). Notice the large amount of free

H-glucosamine in the

HUAEC sample.

Perlecan from Endothelial Cells 31

3.6. Ethanol Precipitation for Concentrating HS

1. To the sample to be concentrated, add 4 volumes of 1.3% CH

COOK in 95% ethanol.

2. Incubate at –20

C for 3–16 h.

3. Isolate the precipitated labeled perlecan by centrifugation in a microfuge at room tempera-

ture for 5 min.

4. Aspirate the supernatant, wash twice (1–5 mL) with salt-free 95% ethanol.

5. The perlecan can be either lyophilized or dissolved in buffer.

3.7. Detection and Quantitation of Perlecan

1. The presence of perlecan in purified and semipurified fractions is monitored using an

ELISA-based screening assay using one antibody that reacts with the protein core (A76)

and another that reacts with the heparan sulfate (10E4). This assay can also be used to screen

for the presence of chondroitin sulfate on the protein core or other co-purifying extracellular

matrix proteins (see Note 12).

2. We have found that the heparan sulfate attached to the protein core of perlecan interfered

with the BCA reaction, resulting in an overestimation of the amount of perlecan present.

Therefore, to avoid these errors, use the dye-binding protein assay, Coomassie Plus Protein

Assay (Pierce), as it is more reliable on the amount of protein core present.

3.8. Electrophoresis

1. Standard SDS-PAGE methodology can be employed to analyze perlecan in gels and West-

ern blots. It is recommended to run 4–15% gradient gels, as the intact perlecan runs at the

Fig. 2. Elution profile of HS derived from immunopurified perlecan using a Superose 6 10/

30 prepacked column. Samples of

H-labeled HS were run through a Superose 6 10/30 column

as described. HUAEC-derived HS is shown by the dashed line, whereas HUVEC-derived HS is

shown by the unbroken line. The V0 of the column was 8 mL (fraction 10), whereas the Vt was

20 mL (fraction 25). The molecular weight of the two types of HS was estimated to be 40 kDa

using previously published standards for chondroitin sulfate (8).

32 Whitelock

bottom of the application wells. The use of 3–8% gradient gels may facilitate the migra-

tion of perlecan into the running gel (see Note 13).

2. SDS-PAGE Gels can be stained with either (see Note 14):

a. 0.25% Coomasie blue in 40% methanol, 50% H

O, 10% CH

COOH.

b. 0.08% Azure A in H

c. 0.1% Alcian blue in 50% ethanol.

d. Or a combination of the above with a AgNO

stain.

3. Composite agarose-polyacrylamide gel electrophoresis (COA-PAGE) can also be

employed to analyze the purified perlecans (see Fig. 3). These gels have the advantage

that the perlecan migrates through the pores of the gel and the distance the molecules

travel depends on the ratio of their mass to their charge. This has the advantage that

perlecans from different sources may be separated on the basis of charge (see Fig. 3). (see

Note 15). They can be blotted to nitrocellulose on a flat bed dry blotter and probed with

antibodies (see Fig. 3).

4. Notes

1. Commercially prepared endothelial cell growth supplement or purified growth factors

such as recombinant human fibroblast growth factors (FGF) or vascular endothelial cell

growth factors (VEGF) may be used in place of the BBE. They are used at a final concen-

tration of 10 ng/mL.

2. Heparin is not required when using either FGF-2 or VEGF. Cells may also be grown

heparin-free by using 15% v/v human serum.

Fig. 3. COA-PAGE immunoblot of perlecan immunopurifed from a HUVEC cell line and

primary HUAEC cultures. Lane 1 represents 3 µg of perlecan immunopurified from a HUVEC

cell line (C11-STH). Lane 2 represents 3 µg of perlecan immunopurified from primary cultures of

HUAECs. Both samples were run through CO-A/PAGE gels and blotted to nitrocellulose. The

nitrocellulose was cut into strips and probed with the anti-protein core antibodies as indicated.

Perlecan from Endothelial Cells 33

3. After 2 weeks of storage at 4°C, the glutamine will have to be re-added at a final concentra-

tion of 2 mM (4 mM for smooth muscle cells). The medium should be discarded after 4 wk.

4. These are prepared essentially as described by McDevitt and Muir (9) (also see

Chapter15).

5. Either

H-glucosamine,

S-Na

, or

S-methionine can be added to the cultures (5 µCi/

mL) to produce biosynthetically labeled perlecan. The choice of labeling reagent depends on

whether you want to label the protein core or the glycosaminoglycan chains.

6. Endothelial cells derived from primary tissue can be maintained at confluence for 1 wk.

The endothelial cell line could be maintained at confluence for longer periods.

7. Biosynthetically labeled perlecan can be prepared using the same methodology as

described for the unlabeled molecule. Its purification can be monitored by sampling

100 µL of each fraction and mixing it with scintillant (Insta-gel; Packard) and counting in

a scintillation counter (LKB 1217 Rackbeta). Radiolabeled perlecan can also be concen-

trated using ethanol precipitation.

8. When using the 3 M MgCl

to elute the column, make the solution fresh before use and

keep the MgCl

stored in a desiccator.

9. Collect the perlecan fractions in siliconized Eppendorf tubes to minimize loss due to

adsorption.

10. The buffers used for the gel filtration step, which may be used instead of the immunoaffinity

step and results in the isolation of perlecan-enriched fractions, may contain 6 M urea as a

dissociative agent. This is particularly pertinent to the isolation of perlecan from tissue sources.

11. When performing the NaBH

/KOH step (overnight at 45°C), choose a screw-capped tube

or cover the tube with parafilm. This prevents the cap popping due to the pressure build up

caused by the release of H

gas. Also, choose a 15-mL tube (for solutions up to 2 mL) so that

during the neutralization step the H

gas that is liberated is allowed to escape without the loss

of the sample.

12. 0.1% casein in PBS is used as both the blocking agent and diluent, due to the fact that it gives

superior backgrounds with the anti-HS antibody, 10E4.

13. If you plan to run gels with a separate stacker, do not remove the stacker before staining or

blotting to nitrocellulose.

14. The SDS must be washed out of the gels with 3 changes in either H

O or 50% ethanol prior

to staining with Azure A, Alcian blue, or silver.

15. These gels can be stained with 0.08% Azure A in H

O by placing them on a sheet of plastic

to act as a support.

Acknowledgments

The author would like to acknowledge Dr. Anne Underwood for discussion and

critical review of the chapter; Penny Bean, Sue Mitchell, and Debbie Lock for excel-

lent technical assistance; and the Australian Government through the Co-Operative

Research Center scheme for funding.

References

1. Hassell, J. R., Robey, P. G., Barrach, H. J., Wilczek, J., Rennard, S. I., and Martin,G. R.

(1980). Isolation of a heparan sulfate-containing proteoglycan from basement membrane.

Proc. Natl. Acad. Sci. (USA) 77, 4494–4498.

2. Paulsson, M., Yurchenco, P. D., Ruben, G. C., Engel, J., and Timpl, R. (1987). Structure

of low density heparan sulfate proteoglycan isolated from a mouse tumor basement mem-

brane. J. Mol. Biol. 197, 297–313.