Figure 8. Size exclusion chromatography and immunoblotting of synuclein proteins.

(A) Alpha, β, or co-incubated mixtures of both proteins were resolved by size exclusion chromatography on a Superose 12 column. Representative chromatograms from a buffer blank, and 20 day in vitro aged α-synuclein, β-synuclein, and co-incubation mixtures are shown. Synuclein proteins eluted as a single peak (Peak 1) in two column fractions at an elution volume of ∼10.89 ml, which corresponds to a molecular weight of ∼57.5 kDa. Peak 2 eluted at ∼15.20 ml, and was an artefact of the in vitro ageing buffer. The migration of proteins of known molecular weight was used to calibrate the column – see inset. (B) Peak column fractions were concentrated, resolved by 1D-PAGE, and stained with colloidal Coomassie blue (upper section), and Western blotted using specific anti-synuclein antibodies (middle section). Beta-synuclein was not immunoprecipitated from either column fractions or aged synuclein reactions when control serum was applied, and β-synuclein antibody did not cross-react with α-synuclein protein. Half of a µg of β-synuclein was similarly blotted to provide a protein standard (lower section, left panel). Beta-synuclein was immunoprecipitated directly from in vitro aged fractions, with the level of β-synuclein recovered confirmed by Western blotting. Parent β-synuclein protein as well as lower molecular weight proteolytic fragments were recovered (marked with arrowheads). Two and a half µg of β-synuclein was similarly blotted to provide a protein standard (lower section, central panel). Beta-synuclein immunoprecipitated from in vitro aged co-incubated mixtures, or from Superose 12 column peak fractions also detected the presence of α-synuclein by Western blotting. Two and half µg of α-synuclein was similarly blotted to provide a protein standard (lower section, right panel). The positions of protein molecular weight standards are also included on Western blots.