Anti syn1 antibody

Interactions between HtrA constructs and α-synuclein were examined by His-mediated pull-down assays. Recombinant HtrA-6His (0.15 mg) was immobilized to 50 μL of Ni-Sepharose resin (GE Healthcare Cytiva, cat: 45002985) and then incubated with 0.15 mg of wild-type α-synuclein at room temperature in assay wash buffer (20mM Tris, 100mM NaCl, 10mM Imidazole, pH 8.0). The incubated mixture was washed five times with wash buffer, and eluted with 500mM imidazole. Protein samples were collected prior to the wash step as ‘Input’. For Western blot analysis, proteins were transferred to nitrocellulose membrane and probed with anti-syn1 antibody (BD Science, Cat 610787). Membranes were imaged using a Li-COR Odyssey FC Imaging system and the amount of protein in ‘input’ and ‘bound’ fractions was determined by densitometry using Image Studio Lite software.

Interactions between HTRA constructs and α-synuclein monomer or fibril were examined by His-mediated pull-down assays. Recombinant HTRA-6His (0.15 mg) was immobilized to 50 µL of Ni-Sepharose resin (GE Healthcare Cytiva, cat: 45002985) and then incubated with 0.15 mg of wild-type α-synuclein at room temperature in assay wash buffer (20 mM Tris, 100 mM NaCl, 10 mM Imidazole, pH 8.0). The incubated mixture was washed five times with wash buffer, and eluted with 500 mM imidazole. Protein samples were collected prior to the wash step as ‘Input’. All samples were then boiled in sample buffer (60 mM Tris, pH 6.8, 5% glycerol, 2% SDS, 4% β-mercaptoethanol) and resolved by SDS-PAGE. For Western blot analysis, proteins were transferred to nitrocellulose membrane and probed with anti-syn1 antibody (BD Science, Cat 610787, 1:1000 dilution). Membranes were imaged using a Li-COR Odyssey FC Imaging system and the amount of protein in ‘input’ and ‘bound’ fractions was determined by densitometry using Image Studio Lite 5.5.4 software.