FIGURE 2.

Western blot demonstrating D-COMP mAb binding to COMP from Gdn-HCl extract of hip articular cartilage. Protein extracts were prepared from osteoarthritic hip cartilage harvested from both an OA lesional site and remote from the lesion in remote OA cartilage. Prior to SDS-PAGE separation on a 10% gel, samples were dialyzed into PBS, protein levels determined, and 5 μg of protein loaded per lane. Samples were separated under standard conditions and transferred to nitrocellulose that was blocked with 3% milk proteins prior to mAb incubation and development with ECL reagents. D-COMP was immunolocated with mAb 6-1A12 specific for deamidated COMP (left) and total COMP was immunolocated with mAb 17-C10 (right). For clarity, the membrane immunostained for total COMP was exposed for a shorter time period (30 s), although the membrane immunostained for D-COMP, due to it lower levels, required a longer exposure (5 min).