Isolation and Purification of α-Synuclein From Human Brain Tissue
This protocol is extracted from research article:
Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking
Front Neurosci, Feb 4, 2021; DOI: 10.3389/fnins.2021.639414

The brain sample used for purifying α-synuclein was provided by the Newcastle Brain Tissue Resource (Newcastle upon Tyne, United Kingdom). This sample was isolated from the cingulate gyrus (cortex) of a healthy control individual [Caucasian female, age 74, post-mortem interval 53 h. Pathology: Braak 0, McKeith none, CERAD (neuritic plaques) none, Braak NFT (TAU) 3]. Consent was obtained from the patient prior to death at the brain collection center. The brain bank approved of the proposal for the use of human tissue in this study, and the IRB at TB’s institution deemed the planned use of this tissue to be appropriate and ethical.

Tissue pieces weighing ∼500 mg were Dounce homogenized with 20 strokes at 2500 rpm in four volumes (weight:volume) Tris-buffered saline (TBS) (20 mM Tris–HCl, 500 mM NaCl, pH 7.5) with a complete protease inhibitor tablet (Sigma-Aldrich, St. Louis, MO, United States). Homogenates were centrifuged for 5 min at 1000 × g at 4°C to remove highly insoluble structures and tissue debris. The resulting supernatants were centrifuged for 30 min at 175,000 × g. The high-speed supernatant was collected, flash-frozen in liquid nitrogen, and stored at 80°C until fractionation. After thawing, the supernatants were fractionated by size and buffer-exchanged into 50 mM ammonium acetate (pH 7.40) using size exclusion chromatography (SEC) with a Superose 12 10/300GL Increase column (General Electric, Boston, MA, United States). Crosslinked samples were run on an 4–12% Bis-Tris gel to determine the molecular species in each sample. The fraction containing the highest level of α-synuclein, as determined by ELISA (SEC fraction 12, corresponding to a molecular weight of ∼60–80 kDa), was split into aliquots and either cross-linked with disuccinimidyl glutarate (DSG) for multimer analysis by Western blotting or left in the native form for axonal microinjections, as described below. Another aliquot of the same sample was immunodepleted of α-synuclein using a monoclonal antibody (Anti-α-Synuclein, clone 2F12, MABN1817 Sigma-Aldrich) for use as a negative control in the synapse experiments.

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