Equine

Ovine

m mm

Fig. 2. Analytical 4-20% SDS PAGE of the same ultracentrifuge samples as examined in Fig. 1. Gels in segments A and D were stained with colloidal Coomassie to visualize the distribution of proteins; proteoglycan species were visualized in B and E by staining with toluidine blue. Replicate gels were also transferred to nitrocellulose (C and F), and G1 domain-containing proteoglycan species were visualized by affinity blotting. The top of the figure illustrates the distribution of protein (31), proteoglycan (as hexuronic acid) (30), and density of the ultracentrifuge samples. The equivalent of 4.1 ^L of original ultracentrifuge fraction was loaded per lane. The migration position of Novex broad-range and See Blue prestained standard proteins are also indicated on the left of the figure.

linear gradient of 0.4-4 M GuHCl in 50 mM Tris-HCl, pH 7, over ~6 bed volumes. Fractions of 10 mL are collected at a flow rate of 10 mL/h. Aliquots of individual fractions are assayed for protein and proteoglycan (as sulfated glycosaminoglycan) (33). The gradient is measured manually using a conductivity monitor.

6. Ice-cold ethanol (3 mL) is added to 1 mL aliquots of individual fractions and the samples shaken at 4°C. The precipitates are collected by centrifugation, washed with 75% ethanol, air-dried and redissolved in SDS PAGE application buffer (0.5 mL).

7. Aliquots (10 ^L/lane) of the ethanol-precipitated samples are examined by 4-20% SDS PAGE and blotted to nitrocellulose, then probed with anti-link protein (Mab 8-A-4). HABPs are identified by affinity blotting. See Fig. 3.

3.3. Isolation of Aggrecan Gl-Proteoglycan Species by Preparative SDS PAGE

1. Freeze-shattered ovine articular cartilage (10 g) is digested with 0.1 mg/mL trypsin in 100mL buffer B for 24 h at 37°C.

2. The tissue residue is collected and washed three times with 50 mL 1M NaCl.

3. The washed trypsinized tissue residue is extracted for 48 h with 100mL buffer A, the extract recovered and dialyzed exhaustively against distilled water and freeze-dried.

4. The freeze-dried material is redissolved in 0.4 M GuHCl and the HABPs isolated by HA affinity chromatography as indicated under Subheading 3.2 but utilizing isocratic elution with 4 M GuHCl to remove the bound HABPs; these are dialyzed against distilled water and freeze-dried (see Note 5).

5. Purified HABP samples are redissolved in 125 mM Tris-HCl buffer, pH 6.8, containing 10% v/v glycerol and 1% w/v SDS application buffer, and heated at 95°C for 5 min.

6. HABP samples from HA affinity (5 mg dry weight) are electrophoresed at 125 V for 90 min in 1.5 mm 4-20% gradient gels using buffer C as running buffer. Chromaphor green stain is added to the upper electrode chamber shortly after commencement of the run in order to visualize protein band separation within the gel during the run (see Note 4).

7. After electrophoresis, the gel is placed in 5% v/v acetic acid for 10 min to improve the visualization of the protein bands. Appropriate regions of the gel are then excised and cut into 1 mm3 pieces.

8. Proteins are electroeluted from the gel pieces using a "little blue-tank electroelution device" (Isco) at 2.5 mA/trap for 2 h at 4°C (23). Each of the electroelution buffer tanks of this device is filled with 90 mL buffer D1. The sample traps are filled with 10 mL buffer D2.

9. The electroeluted samples are examined by analytical SDS PAGE and Western and affinity blotting to confirm the identities of the purified HABPs (see Notes 6 and 7). See Fig. 4.

3.4. Aggrecan MMP-3 Digestions: Detection of G1 Domain Containing Proteoglycan Species by Western and Affinity Blotting

1. rProMMP-3 samples (24) are activated in 1 mM APMA in buffer E for 3 h at 37°C, then applied to HiTrap desalting columns (Pharmacia) to remove the APMA and diluted to a concentration of 40 ^g/mL by protein assay (31).

2. D1 aggrecan samples (0.8 mg/mL) are dissolved overnight at 4°C in buffer E.

3. Proteoglycan samples ([0.4 mg/mL] final) are digested at 37°C for up to 18 h with 20 ^g/mL final MMP-3.

4. Aliquots (0.2 mL) of the digests are removed after 0, 4, and 18 h and added to 50 ^L of 20 mM EDTA to stop enzymatic activity and stored at -20°C until required.

Fig. 3. (A) Fractionation by hyaluronan (HA) affinity chromatography (32) of hyaluronan-binding proteins (HABPs) in an extract of trypsinized bovine nasal cartilage, showing the distribution of proteoglycan species (as sulfated GAG) (33) and protein (31) and visualization of HABP pools 1 and 2 by affinity blotting (inset). (B) Aliquots of individual fractions were also examined by 4-20% gradient SDS PAGE, blotted to nitrocellulose, and link protein species identified using Mab 8-A-4 and HABPs identified by affinity blotting. The migration positions of Novex See Blue prestained standards are also indicated on the left-hand side of the figure.

Fig. 3. (A) Fractionation by hyaluronan (HA) affinity chromatography (32) of hyaluronan-binding proteins (HABPs) in an extract of trypsinized bovine nasal cartilage, showing the distribution of proteoglycan species (as sulfated GAG) (33) and protein (31) and visualization of HABP pools 1 and 2 by affinity blotting (inset). (B) Aliquots of individual fractions were also examined by 4-20% gradient SDS PAGE, blotted to nitrocellulose, and link protein species identified using Mab 8-A-4 and HABPs identified by affinity blotting. The migration positions of Novex See Blue prestained standards are also indicated on the left-hand side of the figure.

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