Sequences from the N-terminal heparin-binding domain and the 10th type III repeat reside together within common subspecies of ~170-kDa synovial fluid fibronectin (FN) fragment. Aliquots of osteoarthritis (OA) sample number 6 (5 μl) were subjected to isoelectric focusing in duplicate pH 4–7 first-dimension (1D) strips, each of which was then subjected to reduced 5% SDS–PAGE. A portion (5 μl) of the sample was also submitted to reduced 1D PAGE in a lane at the edge of each of the two second-dimension gels. After incubation with anti-N-terminal heparin-binding domain mAb followed by HRP-conjugated secondary antibodies, similar enhanced chemiluminescence (ECL) staining patterns were obtained for the resulting two membranes (a, c) after a film development time of 1 minute. Specifically, two major bands were evident in the 1D lane, representing ~200+ (upper arrow) and ~170-kDa (lower arrow) species. Additionally, three major 'spots' (denoted by three vertical arrows), consistent with ~170-kDa species (brackets facing upward), were evident as a nearly horizontal array in the cathodic half of each membrane, approximating the point of migration of the corresponding species within the 1D lane. A cluster of staining with migration approximating that of the ~200+ kDa band was also evident in each membrane (brackets facing downward). The membranes were stripped of antibodies for 30 min at 50°C in 6.25 mM Tris pH 6.7 containing 100 mM β-mercaptoethanol and 2% SDS, then washed in TBST and reblocked with blotto. One was stained with mAb A2C2 diluted in blotto (panel B), whereas the other was incubated in blotto alone (panel D). After incubation with HRP-conjugated secondary antibodies, both membranes were again subjected to ECL development and film exposure for 10 min. Staining was evident in the membrane that had been incubated sequentially with anti-CBD mAb followed by secondary antibodies (b), but not in the membrane exposed only to secondary antibodies (d). When the films shown in (a) and (b) were overlaid using membrane 'edge staining' as a guide, the three ~170-kDa spots present in (a) were found to occupy indistinguishable spatial positions as compared with the corresponding spots evident in (b). In comparison with the anti-N-terminal mAb, mAb A2C2 produced preferential staining of the ~200+ in comparison with the ~170-kDa species.