Cell lysis and immunoblotting for protein and phospho-protein quantification

Quickly wash cells twice with ice-cold PBS. After the second wash, tilt the dish and completely aspirate all residual PBS.

Immediately add ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer. Place cells On ice.

Scrape cells off the dish using a cell lifter.

Transfer the cell lysate to an Eppendorf tube On ice.

Leave lysates On ice for 0h 20m 0s to allow for efficient lysis.

Centrifuge for 0h 10m 0s at 17000x g,0h 0m 0s and 4°C.

Discard pellet and use clarified supernatant to determine protein concentration by BCA assay following the manufacturer’s instructions, performing all measurements in triplicates.

Add 100µL ß-mercaptoethanol to900µL of 4x Protein Loading Buffer and mix well.

Add complete 4x Protein Loading Buffer to cell lysates, mix well, and boil for 0h 5m 0s at 95°C.

Load samples onto 8% (for LRRK2 protein) to 15% (for PPM1H and Rab proteins) acrylamide gels alongside Chameleon Duo pre-stained protein ladder (LI-COR).

Start electrophoresis at 80 V for 0h 20m 0s, then increase to 120 V and electrophorese until orange dye runs out.

Activate Immobilon-FL PVDF membrane by submerging in methanol for 0h 0m 30s- 0h 1m 0s.

Wash in ddH₂O and equilibrate in transfer buffer.

Soak sponges in methanol, wash in ddH₂O and equilibrate in transfer buffer.

Equilibrate filter paper in transfer buffer.

Assemble blotting sandwich.

Carefully remove any air bubbles between layers using a roller.

Fill transfer tank with ice-cold transfer buffer.

Place transfer system On ice.

Transfer proteins from gel onto PVDV membrane at 100 V for 1h 15m 0s.

After wet-tank transfer, let membrane dry completely for at least 1h 0m 0s at Room temperature.

Rehydrate membrane for 0h 1m 0s in 100% methanol, then wash 0h 5m 0s in 1x TBS.

Incubate for 0h 5m 0s in Revert total protein stain (LI-COR) while gently shaking at Room temperature.

Wash twice with Revert wash solution, then rinse in ddH₂O and image membrane on ODYSSEY CLx imaging system.

Remove Revert total protein stain by incubating membrane in Revert reversal solution for 0h 10m 0s at Room temperature while gently shaking.

Rinse membrane with ddH₂O.

Block membrane for 0h 5m 0s in Everyblot Blocking Buffer (Bio-Rad) at Room temperature.

Dilute primary antibodies in Everyblot Blocking Buffer and incubate at 4°C 0h 5m 0s.

Wash membrane in 1x TBS + 0.1% Tween-20 (TBS-T) at Room temperature (4 washes, 0h 5m 0s each).

Dilute secondary antibodies 1:20,000 in Everyblot Blocking Buffer and 0.02% SDS.

Wash membrane in TBS-T at Room temperature (4 washes, 0h 5m 0s each).

Rinse membrane with TBS (no detergent), then image on ODYSSEY CLx imaging system. Quantify signal intensity using Image Studio Software.