Additional file 1: Figure S1. of A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Principle and protocol of the AlphaLISA technique. In A Streptavidin-coated Donor-beads are bound to biotinylated antibodies. Acceptor-beads are directly coupled to antibodies. When both antibodies bind to an analyte, the beads are brought into close proximity. Donor-beads are excited at 680 nm and produce singlet oxygen molecules triggering a chemical reaction in the close-by Europium Acceptor-beads resulting in an emission of light at 615 nm. In B the protocol of a no-wash AlphaLISA is depicted. (PDF 315 kb

Additional file 2: Figure S2. of A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Quantification of S129 phosphorylation of human alpha-synuclein recombinant protein. An isoelectric focusing gel pH range 5–8 (A) was used to separate pS129 from non-phosphorylated h-asyn by loading recombinant h-asyn and pS129 h-asyn protein side by side. In B, blots were probed for total h-asyn (syn-1) or pS129 h-asyn (11A5) to determine pS129 h-asyn band. Quantification areas are indicated on total h-asyn blots by dashed boxes. This assay was run independently on three occasions, which yielded estimates of percent phosphorylation of h-asyn at S129 site to be 28.4, 29.5, and 26.0 %, respectively, with an average and standard deviation of 28.0 ± 1.8 %. (PDF 339 kb