Anti alpha synuclein

hαSyn (1 mg/ml) was incubated with hMPO (20 nM), glucose oxidase (GO, 20 ng/ml) and glucose (100 μg/ml) as a source of H₂O₂ and either sodium nitrite (500 μM) or potassium cyanate (340 μM) for 6 h at 37°C. Proteins were fractionated by SDS-PAGE (4%–12%). Proteins were then transferred to nitrocellulose and probed with the respective antibodies: anti-alpha synuclein (1:1,000, BD), anti-nitrated tyrosine (1:1,000, Millipore), anti-carbamylated αSyn (1:1,000, described here). Blots were incubated in 5% Blotto in PBST for 1 h and then incubated in PBST containing 1% BSA with the appropriate antibody for 2 h at RT. The membranes were washed for 1 h in PBST at RT. Membranes were then incubated with HRP-labeled secondary antibodies in PBST + 1% BSA for 1 h. Following this incubation the blots were washed in PBST and then incubated with ECL reagent and exposed to X-ray film. For analysis of αSyn by liquid chromatography tandem MS (LC-MS/MS), samples were treated as above but the gel was stained with Commassie blue and the band containing the αSyn monomer was excised and analyzed for post-translational modifications.