One hundred corneas were extracted in 4 M guanidine hydrochloride for 12 hours at 4°C in the presence of protease inhibitors (0.05 M sodium acetate, 0.01 M disodium EDTA, 0.1 M 6-amino hexanoic acid, 0.005 M benzamidine HCl, and 0.5 mM PMSF [pH 5.8–6.8]). Supernatants were collected, and the tissue was subjected to repeat extraction for a further 12 hours at 4°C. Pooled extracts were dialyzed into 6 M urea in 0.1 M Tris-HCl, 0.1% CHAPS (3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate). The crude PG extract was applied to a diethylaminoethyl (DEAE) Sepharose fast flow anion exchange chromatography resin (Amersham Pharmacia Biotech, Piscataway, NJ). Bound PGs were eluted with 1.5 M NaCl, then dialyzed into double-distilled water (Milli-Q; Millipore, Bedford, MA). Dimethylmethylene blue analysis revealed 60 μg GAG in the preparation. The sample was freeze dried and reconstituted in 60 μL 0.1 M Tris acetate (pH 6.8). A portion of the sample (30 μL) was digested with 0.01 U keratanase (EC 3.2.1.103; MP Biomedicals [formerly ICN Biomedicals], Irvine, CA), and 0.01 U chondroitinase ABC (EC 4.2.2.4; Sigma-Aldrich, Poole, UK) per milligram GAG.
Digested and undigested samples were mixed with an equal volume of 2× SDS buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, and 0.01% bromophenol) and 10% β-mercaptoethanol. The samples were reduced by boiling for 5 minutes, and subsequently 3 μg of GAG was loaded onto 4% to 20% Tris glycine gradient gels (Invitrogen, Renfrew, UK) for Western blot analysis using the monoclonal antibodies 5D4, 1B4, 4D1, 1B5, 2B6, and 3B3. These antibodies are well characterized
25 26 27 28 and are specific for KS or CS/DS-GAGs on PGs. They recognize discrete epitopes defined by sulfation patterns on the disaccharide chains, some of which require exposure by partial predigestion with purified enzymes (summarized in
Table 1 ). Chondroitinase and keratanase digestion were normally used in tandem to generate the immunoepitope and facilitate antibody penetration.
Antibody 2B6 can also be used to investigate the presence of both DS- and CS-GAG chains on mouse corneal CS PGs by differential digestion with chondroitinase ABC and AC II enzymes. This is possible because chondroitinase ABC digestion generates 2B6-reactive neoepitope from both CS- and DS-GAG chains, whereas chondroitinase AC II does not digest DS and thus only generates the neoepitope C-4-S “stub” from CS chains. Chondroitinase AC II (AC II Arthro 4.2.2.5; Seikagaku Corp., Tokyo, Japan), digestion of corneal extracts was as described earlier for chondroitinase ABC.