Fig. 2. PEPperMAP epitope mapping.

a Either 1 μg/ml (blue line) or 10 μg/ml (red line) of IgG 306C7B3 were incubated with membranes spotted with 140 different 15mer peptides (each peptide printed in duplicate to be present in the top and bottom part of the array), representing the full human α-synuclein sequence with each peptide having a 14 amino acid overlap with the preceding peptide with the human α-synuclein sequence being elongated by neutral GSGSGSG linkers to avoid truncated peptides. Inset shows microarray stained with 10 μg/ml 306C7B3 (green: 306C7B3 signal, red: HA signal). Outer spots correspond to control peptides used to orient the microarray. Some cross-reactivity against the control peptides is observed for 306C7B3 at this high antibody concentration. Figure shows signal intensity obtained for individual peptide spots, identifying the epitope of 306C7B3 as being “YQDYEPE” (amino acids 133–139 of full-length human α-synuclein) located in the C-terminus of α-synuclein with overall low binding intensity toward the microarray spotted α-synuclein peptides. b Similar approach to determine the epitope of Syn1 at a concentration of 1 μg/ml (blue line, red: Syn1 signal, green: HA signal), identifying the epitope of Syn1 as being “AATGFVKK” (amino acids 90–97 of full-length human α-synuclein). Note that the exemplified peptide sequences do not reflect all tested 15mer peptides for better visualization—identical peptide arrays were used for the analysis of the different antibodies.