KS-PGs run as smears on Western blot analysis after SDS-PAGE because they are heterogeneous in size given the variations in size and number of the KS chains on the PG core protein. Thus, 5D4 immunostaining detects two major smears, an upper one at approximately 200 kDa and a lower one at approximately 75 kDa (
Fig. 1A , lane 1). Predigestion with keratanase reduces the intensity of the upper smear and causes the lower smear to move to approximately 50 kDa, with an additional smear at approximately 37 to 27 kDa (
Fig. 1A , lane 2). The smaller molecular weight smears likely result from KS-PG catabolites in the corneal extract. Keratocanase digestion on the blot (
Fig. 1 , lane 3), before immunolocalization with 5D4, produces essentially the same staining pattern as the undigested Western blot (
Fig. 1 , lane 1). This means that keratanase digestion has reduced the length of the KS chains on the KS-PGs in the corneal extract and that some of the 5D4 epitope is present on the truncated KS-GAG chains still attached to the PG core proteins. Immunostaining with BKS-1 was negative on the Western blot of native corneal KS-PGs that had not been predigested with keratanase (
Fig. 1 , lane 4), but strong immunostaining was detected with the KS-PGs after keratanase pretreatment (
Fig. 1 , lane 5). These smears now migrated at approximately 50 to 75 kDa, with some weaker staining of KSPGs at approximately 200 kDa and even weaker reactivity with KS-PGs migrating at 27 to 37 kDa. This result indicated that the KSPGs migrating at approximately 200 kDa and 27 to 37 kDa had little KS containing monosulfated
N-acetyl-lactosamine disaccharides that would allow keratanase cleavage of the KS GAG chains and generation of the BKS-1 neoepitope. Immunostaining of the native corneal KSPGs after keratanase treatment on the blot (
Fig. 1 , lane 6) showed essentially the same staining pattern as that seen for the immunostaining with 5D4 under similar conditions (
Fig. 1 , lane 3). This result indicated that both species/smears of KS-PGs, found at approximately 200 kDa and approximately 75 kDa (
Fig. 1 , lanes 1, 3, 6), contained some KS that was digestible with keratanase and the production of the BKS-1 neoepitope.