Immunoblot of selected scFvs with purified intact TSP and TSP digests. Bacterial supernatants containing expressed soluble scFv (TSP-A10, -C5, -C6, -D2, -E7, and -E9) were incubated with (A) purified human platelet TSP or (C-D) TSP digested with thermolysin in the presence of CaCl₂ (Th/+), chymotrypsin in the presence of CaCl₂ (Ch/+), or chymotrypsin in the presence of EDTA (Ch/-) resolved by PAGE under reducing (A,C) or nonreducing (D) conditions and transferred to PVDF membrane. Bound scFv was detected by anti–c-myc mAb 9E10 as described in “Materials and methods.” (B) Schematic illustration of TSP monomer with selected domains as described in the text. HBD indicates heparin-binding domain; PC, procollagen-like region; type 1, type 1 repeats; type 2, type 2 repeats; Ca⁺⁺ Binding, Ca++-binding domains or type 3 repeats; CBD, cell-binding domain; S, site of cysteines that disulfide link TSP chains to form trimer; and S-S, disulfide bond in C-terminal region. The approximate locations of the 25-kDa, 140-kDa, 120-kDa, 18-kDa, and 70-kDa proteolytic fragments as identified by amino-terminal sequencing and size^26,28,33  are indicated by arrows. The 25-kDa fragment is a monomer,²⁶  the 70-kDa, 120-kDa, and 140-kDa fragments are trimers,33 and the 18- to 20-kDa C-terminal fragment remains disulfide linked to the 120-kDa fragment.¹⁷