Western Blot

Prepare the appropriate amount of RIPA buffer with Protease and Phosphatases Inhibitor Cocktail (Roche) and maintain On ice

Collect cells/organoids in PBS On ice in an eppendorf and spin down 500rpm,4°C

Add appopriate amount of lysis buffers (1:50 v/v proportion between cell pellet and lysis buffer) to the pellet and pipette up and down

For organoid samples use an homogeniser

Incubate samples for 0h 30m 0s On ice . Spinning every 0h 10m 0s

Centrifuge at 15000x g,0h 0m 0s for 0h 10m 0s at4°Cand collect the supernatant in a new tube.

If SDS fraction is desired, add 2Mass / % volume SDS to RIPA buffer and resuspend pellet from step 5.

Sonicate the sample a 3 times for0h 0m 10s to ensure fragmentation of genomic DNA released during lysis and consequently reduce viscosity

Quantify protein concentration.

Mix 20-30µg of protein with Laemmli's loading buffer

Denaturate the proteins by incubation at 95°C for 0h 5m 0s , spin briefly before loading

Load pre-cast gel into Western Bloat apparatus and fill with Running Buffer (BioRad).

Load samples and protein ladder into gels, Run the gel at 120V for 0h 45m 0s to ensure protein separation.

Transfer proteins into a PVDF membrane (previously activated with methanol and hydrated in ddH2O)

Remove membrane from transfer and place into a box with blocking buffer: 5% BSA In TBS-T (20mM Tris-HCl, 150mM NaCL pH8, 0.1% Tween20). Block for 1h 0m 0s

If alpha-synuclein is to be blotted, fix the membrane with 4% PFA in PBS for 0h 30m 0s prior to blocking

Once blocked, sequentially probe the membrane for antibody staining and detection. Antibody dilution might need optimization.

Prepare primary antibody in 5% BSA in TBS-T and left incubating 0h 30m 0s at 4°C

Wash membranes 3x 0h 10m 0s in TBS-T and then incubated for 1h 0m 0s at Room temperature with the appropriate HPR-tagged secondary antibody.

Membrane was washed 3x 0h 10m 0s in TBS-T

Mix EZ-ECL solution 1:1 (v/v) and incubate on the membrane for 0h 1m 0s

Image membranes using a Licor Odyssey Chemioluminescence Imager.