Anti α synuclein antibody

Recombinant α-synuclein solution was mixed in equal amounts with a sample-loading buffer. After denaturation by boiling at 100 °C for 5 min, samples were loaded onto a 4‒20% SDS-polyacrylamide gel, separated electrophoretically, and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Each lane contains the same amount of protein (1.5 ug). After blocking with non-fatty milk, the membrane was incubated with anti-α-synuclein antibody (Sigma) and horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). Immunodetection was performed using the ECL Western blotting detection system (GE Healthcare).

GST-CaBP1 protein (100 ng/µL) was added in equal amounts to αSNo containing wild type recombinant α-synuclein (10 µM) and dopamine (100 µM) and incubated for 1 h at 37 °C. The mixture was incubated with or without anti-CaBP1 antibody (Sigma) for 1 h at 37 °C followed by incubation with Protein G sepharose 4 Fast Flow (GE Healthcare) for 1 h at 4 °C with gentle shaking. The beads were precipitated by centrifugation and washed four times with an excess volume of Tris-buffered saline containing 0.1% Triton X-100. Proteins bound to beads were eluted by boiling in a sample-loading buffer. Western blotting was performed as described above, except that anti-α-synuclein antibody (Sigma) and anti-GST antibody (Nacalai Tesque, Kyoto, Japan) were used.