Journal: Molecular Neurodegeneration
Article Title: Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis
doi: 10.1186/s13024-016-0145-9
Figure Lengend Snippet: Anti-SOD1 int antibody exclusively recognizes soluble disulfide-crosslinked SOD1 oligomers in vitro. a The antibodies were tested for their specific reactivities to soluble disulfide-crosslinked oligomers (black filled bars) over Cu,Zn-SOD1(WT) S-S (open bars) and E,E-SOD1(A4V) S-S (gray filled bars) by indirect ELISA. Antisera were either affinity-purified with the corresponding peptides (w/o absorption) or first absorbed with SOD1(WT) S-S and then affinity-purified with the peptides (w/ absorption). Anti-SOD1 48–53 antibody obtained after the absorption exclusively reacted with soluble disulfide-crosslinked oligomers and called anti-SOD1 int antibody. b - d The reactivities of b anti-SOD1 int , c USOD-like, and d SEDI-like antibody were examined with indirect ELISA. Several forms of SOD1 (WT, A4V, G37R, G85R) with a distinct metallation/disulfide status, soluble disulfide-crosslinked oligomers and insoluble amyloid-like aggregates were prepared and fixed on an ELISA plate. The ELISA signal was represented as a ratio against that obtained using BSA. Three independent experiments were performed to estimate error bars (standard deviation). Fixation of equal amounts of SOD1 proteins on each well of an ELISA plate was confirmed by ELISA using polyclonal anti-SOD1 antibody (FL-154, Santa Cruz Biotechnology), which is shown in Additional file : Figure S5
Article Snippet: After six washes with TBS-T, either antibody purified in this study, polyclonal anti-human SOD1 (FL-154, Santa Cruz Biotechnology), USOD (#SPC-205, StressMarq Bioscience), or SEDI (#SPC-206, StressMarq Bioscience) antibody was added as a primary antibody (0.2 μg/mL) and incubated for an hour at room temperature, which was then followed by secondary antibody with horseradish peroxidase (goat anti-rabbit IgG, 1:1,000; Thermo Scientific) for an hour at room temperature.
Techniques: In Vitro, Indirect ELISA, Affinity Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation
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