Perform extraction and detection of Triton-soluble (T-sol) and Triton-insoluble (T-insol) alpha-synuclein as described in Stojkovska and Mazzulli(2021).

Lyse individual organoids in 1% Triton X-100 extraction buffer supplemented with 1X PIC, 50millimolar (mM) NaF, 2millimolar (mM) NA₃VO₄ and 0.5millimolar (mM) PMSF.

Extraction buffer

A	B
Triton X-100	1%
NaCl	150 mM
glycerol	10%
HEPES pH 7.4	25 mM
EDTA	1 mM
MgCl2	1.5 mM

Homogenize samples with a pestle and incubate on a platform shaker in an ice-water slurry for 0h 30m 0s, followed by three freeze/thaw cycles and ultracentrifugation at 100000x g,4°C.

Collect the supernatant (Triton-X Soluble fraction).

Wash the remaining pellet in Triton X-100 extraction buffer followed by another ultracentrifugation at 100000x g,0h 0m 0s.

Resuspend the pellet in 2% SDS buffer containing 50millimolar (mM) Tris, 7.4 and 1X PIC, boil it for 0h 10m 0s at 100°C (Triton-X insoluble Fraction) and label the T-insol fraction.

Sonicate Tx-Insoluble samples for 0h 10m 0s at 30% power, 20C in a cup horn sonicator (Qsonica-Q700), and then boil them again for 0h 10m 0s at 100°C.

Ultracentrifuge Tx-Insoluble samples at 100000x g,21°C.

Collect the supernatant (SDS-soluble fraction).

Detect protein concentrations using a BCA assay and load 30µg of total protein for each condition.