6533b882fe1ef96bd12d9d7d

RESEARCH PRODUCT

Additional file 6 of The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

subject

description

Additional file 6: Fig. S4. Detection of HAS-7 by Western blot. (a) Antigenic peptide competition demonstrates the specificity of the HAS-7 antibody. A Western blot for tissue lysates as in Fig. 3a was performed using primary antibody solution with (right panel) or without (left panel) 1 mg/ml of the antigenic peptide used for generating the HAS-7 antibody. The HAS-7 peptide effectively reduces the detection of specific bands at ~ 40 and 70 kDa. (b) Ni-NTA affinity purified recombinant HAS-7. Separation by 12% SDS-PAGE was followed by staining with Coomassie brilliant blue (left) or transfer to PVDF and immunodetection (right) using the Penta-His-antibody as described above. For each lane 1.8 μg of recombinant HAS-7 protein eluted with 250 mM imidazole were applied. M, marker proteins as indicated. (c) Dilution series of recombinant HAS-7 and native HAS-7 protein in HL detected with anti-HAS-7 antibody show a double band at 42 kDa for recombinant HAS-7 while in the HL two double bands migrating at 38 kDa and 42 kDa, respectively, are detectable. This heterogenous pattern likely represents a mixture of immature and posttranslationally modified forms of HAS-7 proteins in the tissue lysate. (d) Upper panel: Western blot detection of HAS-7 in full hydra lysate and elution fractions after cation exchange chromatography as indicated. Fractions 15-21 were pooled as they showed a subcritical protein concentration (