Brain synaptic and mitochondrial human α-synuclein were resolved mainly as a monomeric protein with truncated species by Native-PAGE and 2-dimensional PAGE (Native/SDS) immunoblotting.

<p>A. An α-synuclein (αSyn)-immunoreactive band of 50–60 kDa plus smaller forms (arrows, bracketed) were detected in sonicated ASOTg (ASO) forebrain SNs (10 µg) by Native-PAGE/immunoblotting. An SN fraction from <i>Snca^−/−</i>mice (KO) served as a negative control. B. As a positive control for monomer conformations, sonicated recombinant human α-synuclein (Rec Human αSyn)(0.2–0.4 µg) was run on a Native gel and detected by immunoblotting (last lane). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa amongst a large smear of bands > than 50 kDa half way up the gel (bracketed). WT and ASOTg cortex mitochondria (mito) and cytosolic (cyt) fractions (20 µg) as well as surgically resected human cortex (20 µg) were resolved on the same blot side by side with recombinant α-synuclein. A major band of 50–60 kDa is resolved but smaller bands were also detected mainly in the ASOTg mitochondrial fraction. C. In a separate Native-PAGE/blotting experiment, a major band of 50–60 kDa was also detected in ASOTg striatum mitochondrial/cytosolic fractions, a cerebellum mitochondrial preparation, recombinant α-synuclein, and five human postmortem midbrain tissues [con (control Case #1), con (control Case #2), PD (Case #4), DLBD/PD (Case #3), AD/DLBD (Case #5). D. Cortex mitochondrial and recombinant human α-synuclein were resolved by two-dimensional PAGE (1-D Native/2-D SDS) and immunoblotting. Native-PAGE isoforms (≥50 kDa) of recombinant α-synuclein were resolved mainly as monomers (mono) close to 20 kDa in size on SDS-PAGE. Similar sized Native-PAGE isoforms (≥50 kDa) observed in ASOTg cortex mitochondrial fractions were also resolved as monomers on SDS-PAGE. Smaller mitochondrial forms (trunc) closer to 15 kDa in size were also detected supporting their COOH-terminal truncation. In a separate 2-D experiment, α-synuclein monomers and truncated forms were also observed in an ASOTg striatum mitochondrial fraction (only 2-D SDS-PAGE shown)(bottom panel).