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Fig. 8 | Molecular Neurodegeneration

Fig. 8

From: Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis

Fig. 8

Misfolded SOD1 in CSF of ALS decreases the affinity toward metal ions. (a) The CSF samples (20 μg of total proteins) were approximately 4-fold concentrated with a centrifugal filter device and then examined for the SOD1 activities with the in-gel assay. Recombinant wild-type SOD1 protein in the holo state was analyzed as a positive control. (b) The CSF samples (20 μg of total proteins, 100 μL of total volume) were first incubated with either (blue bars) 100 μM CuSO4 or (red bars) 100 μM ZnSO4 at 4 °C overnight and then analyzed by sandwich ELISA with C4F6 and Pan-SOD1 as capture and detection antibodies, respectively. As a control, the CSF samples incubated at 4 °C overnight without addition of any metal ions (black bars) were also examined. As described in the Methods, absorbance values at 490 nm measured in the ELISA were substituted into equations fitted to standard curves (Additional file 3: Figure S2), and apparent amounts of SOD1 were thereby estimated and plotted as SOD1 (ng/20 μg of total CSF proteins). The experiments were performed in triplicate to estimate error bars (standard deviation)

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