Ser(P)-129 α-syn antibodies and the antibody raised to total alpha synuclein were commercially available and purchased from Abcam (EP1536Y,81a, MJF-R13), Wako USA (pSyn#64), and BD Bioscience (Syn1). Vendors report that the Ser(P)-129 α-syn monoclonal antibodies were raised against synthetic human α-synuclein peptide spanning 124–134 region with a phosphorylated Ser 129 residue. This peptide sequence was originally constructed by Fujiwara et al, and used in their seminal work that led to the discovery of extensive α-syn phosphorylation at Ser 129 reside in Lewy bodies (Fujiwara et al., 2002 (link)). Monoclonal antibodies developed against synthetic α-syn aa 124–134 (phospho 129) peptide were validated using other antibodies that label Lewy bodies in post-mortem brain sections (Baba et al., 1998 (link); Fujiwara et al., 2002 (link)).
Clone EP1536Y is a monoclonal IgG antibody raised in rabbit and may be suitable for use in immunoblotting and immunohistochemistry (Perez-Revuelta et al., 2014 (link); Prusiner et al., 2015 (link); Tuttle et al., 2016 (link); Arawaka et al., 2017 (link)). Clone 81a is mouse IgG2a also used in immunoblot and immunohistochemistry applications (Tuttle et al., 2016 (link); Dieriks et al., 2017 (link)). MJF-R13 is a rabbit IgG monoclonal antibody used in immunoblotting experiments (Brahmachari et al., 2016 (link)). Clone pSyn#64 is mouse IgG1 suitable for immunoblotting and immunohistochemistry (Jiang et al., 2016 (link)). Clone Syn1 is mouse IgG1 raised to rat α-syn peptide aa 15–123, has not been further mapped within that peptide, but has been previously evaluated for specificity (Perrin et al., 2003 (link)). For additional primary antibody information and secondary antibody information see Table 1. Optimal secondary concentration was determined by serial dilution starting with the vendor recommendations. pS129-α-syn antibody concentration was confirmed using a BCA Protein Assay Kit (Pierce) with a γ-globulin protein standard, and all antibodies diluted to working stock concentrations of 0.1 mg mL−1.