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letter
. 2014 Apr;99(4):e58–e60. doi: 10.3324/haematol.2013.100685

Figure 1.

Figure 1.

Epitope mapping of affinity-purified anti-ADAMTS13 IgG antibodies by immunoprecipitation and Western blotting. (A) Schematic domain representation of rADAMTS13 and variants used. FL: Full length; MDT: metalloprotease, disintegrin and 1st thrombospondin type 1 repeat domains; MDTCS: metalloprotease, disintegrin, 1st thrombospondin type 1 repeat, Cys-rich and spacer domains; T2C: 2nd – 8th thrombospondin type 1 repeat and two CUB domains; CUB: two CUB domains. (B) Detection of purified rADAMTS13 and variants (0.1 μg/lane) by SDS-PAGE and Western blotting with mouse anti-V5 IgG (1:5,000) (Invitrogen, Carlsbad, CA, USA) and IRDye 800-labeled goat anti-mouse IgG (1:10,000) (LICOR Bioscience, Lincoln, Nebraska, USA). (C–F) Binding pattern of the IgG preparations isolated from patient 1 (C), patient 2 (D), patient 3 (E) and an HD pool (F). 0.5 μg of the distinct IgG preparations and near equivalent amounts (0.5 μg) of either full length rADAMTS13 or variants were incubated with 30 μL protein G-conjugated Sepharose beads (GE Healthcare, Uppsala, Sweden) in 200 μL binding buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Triton X100 and 0.1% Tween20 containing 0.1% casein, 5 mM EDTA, and 0.2% protease inhibitor cocktail from Sigma) overnight at 4°C with rocking. After being washed, the antibody-antigen complex was eluted from the protein G beads with 30 μL 5X SDS buffer and heated for 10 min at 100°C. The bound ADAMTS13 and variants were determined by Western blotting as described in B. FL-IgG (incubation of FL ADAMTS13 in the absence of IgG) and FL+anti-V5 (incubation of FL ADAMTS13 with mouse anti-V5 IgG) refer to the negative and positive control, respectively. FL(1/5) refers to FL ADAMTS13 (0.1 μg) loaded directly on the gel. The loaded negative control “FL-IgG” in D and E refers to 1/5th of the amount loaded in panels C and F. The discernible 75 kDa (1), 50 kDa (2) and 37 kDa (3) bands refer to degradation products of ADAMTS13 constructs that still have the V5 at their C-terminus. A non-specific 25 kDa double band discernible in all lanes is indicated by an asterisk.