Gel Imaging and Product Quantitation see Figs

1. After completion of the electrophoresis, one gel cassette at a time is removed from the tank. The outside of the glass plates as well as the loading wells are extensively washed with distilled water to remove running buffer and excess unreacted AMAC reagent (see Note 11).

2. The sample wells and the stacking gel are covered with light impermeable tape, and the gel cassette is placed on the transilluminator light box (see Note 12).

3. The gels are viewed and the images captured and stored in TIFF file formats.

4. Using standard image analysis software, an average pixel density per picomole of the A-disaccharide standards (based on the glucose standard) is determined. From that, A-disaccharide contents and composition in experimental samples are calculated (see Notes 9, 13-15).

5. A typical FACE gel of the chondroitinase ABC digestion products obtained from gly-cosaminoglycans prepared from fetal and adult bovine cartilages and adult human cornea

Fig. 4. Quantitation of a fluorotagged saccharide after FACE separation using pixel density measurements. (A) The fluorescent image and the inverted image used for determination of pixel density values of various concentrations of fluorotagged galactose. (B) Double log plot of pixel density per band vs. picomoles of saccharide per band, with near linearity between -20-200 pmol of saccharide.

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Fig. 5. FACE analyses of A-disaccharide standards. Two separate preparations of 1 nmol each of A-di-HA, A-di-0S, A-di-6S, A-di-4S, and A-di-E (based on the quantity of disaccharide obtained from the supplier) and 1 nmol of glucose were fluorotagged and 10% portions of each reaction mixture (panel A, L1 and L2) were analyzed by FACE (see Subheading 3.5. in text for details).

Fig. 6. FACE analyses of chondroitinase ABC-digested tissue glycosaminoglycans. 10 ^g of glycosaminoglycans prepared from fetal bovine (FB) or adult bovine (AB) cartilages and from human cornea (HC) were digested with chondroitinase ABC, fluorotagged, and analyzed by FACE as outlined in the experimental procedure. The left-hand panel shows a short-exposure image that was used for quantitation of the major disaccharide products (A-di-0S, A-di-6S, and A-Di-4S). The right-hand panel shows a longer exposure that was used to visualize and quantitate minor products, such as A-di-HA, and the nonreducing terminal monosaccharide galNAc4S (indicated by white arrows). Nonspecific fluorescent bands from the reagents themselves are indicated by (*). A standard curve for product quantitation by pixel densities (B) was generated from the standard A-disaccharides and glucose shown in lane S (ranging from 25-270 pmol) and at a 1:4 dilution in lane S'.

Fig. 6. FACE analyses of chondroitinase ABC-digested tissue glycosaminoglycans. 10 ^g of glycosaminoglycans prepared from fetal bovine (FB) or adult bovine (AB) cartilages and from human cornea (HC) were digested with chondroitinase ABC, fluorotagged, and analyzed by FACE as outlined in the experimental procedure. The left-hand panel shows a short-exposure image that was used for quantitation of the major disaccharide products (A-di-0S, A-di-6S, and A-Di-4S). The right-hand panel shows a longer exposure that was used to visualize and quantitate minor products, such as A-di-HA, and the nonreducing terminal monosaccharide galNAc4S (indicated by white arrows). Nonspecific fluorescent bands from the reagents themselves are indicated by (*). A standard curve for product quantitation by pixel densities (B) was generated from the standard A-disaccharides and glucose shown in lane S (ranging from 25-270 pmol) and at a 1:4 dilution in lane S'.

is shown in Fig. 6. Two images representing different exposure times were used to visualize and quantitate the abundant A-disaccharides and minor products, respectively. The results are summarized in Table 1.

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