Sequential Extraction of Alpha-Synuclein

Sequential extraction of αSYN from brain tissue was performed as previously described [37 (link)]. In short, flash-frozen brain tissue samples were homogenized with a PreCellys Evolution (VWR, Stockholm, Sweden) in ice-cold Tris-buffered saline (TBS) supplemented with cOmplete protease inhibitor (Roche, Mannheim, Germany) and PhosStop phosphatase inhibitor (Sigma, Gillingham, UK) at a 1:10 w/v ratio. Next, αSYN species of decreasing solubility were extracted with TBS with 0.5% Triton X-100 (TBS-T) at 16,000× g and 70% formic acid (FA) at 100,000× g.

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Roshanbin S., Julku U., Xiong M., Eriksson J., Masliah E., Hultqvist G., Bergström J., Ingelsson M., Syvänen S, & Sehlin D. (2022). Reduction of αSYN Pathology in a Mouse Model of PD Using a Brain-Penetrating Bispecific Antibody. Pharmaceutics, 14(7), 1412.

Publication 2022

PreCellys Evolution cOmplete protease inhibitor PhosStop phosphatase inhibitor

Brain Cold Evolution Formic acid Frozen Phosphatase Protease inhibitor Saline Tissue Triton x 100

Corresponding Organization : Uppsala University

Other organizations : Uppsala University Hospital, National Institutes of Health, University of Toronto, University Health Network

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Variable analysis

independent variables

Sequential extraction of αSYN from brain tissue
Homogenization of flash-frozen brain tissue samples with a PreCellys Evolution in ice-cold Tris-buffered saline (TBS) supplemented with cOmplete protease inhibitor and PhosStop phosphatase inhibitor at a 1:10 w/v ratio
Extraction of αSYN species of decreasing solubility with TBS with 0.5% Triton X-100 (TBS-T) at 16,000× g
Extraction of αSYN species of decreasing solubility with 70% formic acid (FA) at 100,000× g

dependent variables

Levels of αSYN species extracted

control variables

Use of cOmplete protease inhibitor and PhosStop phosphatase inhibitor in the homogenization buffer
Centrifugation speeds (16,000× g and 100,000× g) for the extraction steps

positive controls

None specified

negative controls

None specified

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