Western blotting for LRRK2 signalling in macrophages

Culture cells as usual in a 12-well or 6-well plate.

Wash cells once in PBS

Add 100 μl per 12-well or 200 μl per 6 well of lysis buffer containing protease and phosphatase inhibitors. Keep samples On ice from now on.

Scrape cells using a cell lifter and transfer lysate to a 1.5 ml Eppendorf tube

Vortex and incubate on ice for 0h 10m 0s.

Clarify lysate by spinning at 17000x g,0h 0m 0s at 4°C for 0h 15m 0s

Transfer supernatant into a fresh 1.5 ml Eppendorf tube and store at -20 °C.

Prepare protein lysates by adding sample loading buffer and reducing agent (eg NuPAGE LDS sample buffer and reducing agent).

Denature samples at 80°C for 0h 8m 0s

Prepare the SDS gel (4-12% Bis-Tris Nu-PAGE gel) by assembling the apparatus, filling the tank with MES running buffer and flushing all the wells with MES running buffer.

Spin down the samples quickly in case of condensation and load wells carefully. Include a broad range molecular weight marker (eg Broad molecular weight ladder, ab116028, Abcam).

Run the gel at 180 V for 0h 35m 0s.

Transfer SDS gel into 20% EtOH while preparing the transfer stack. Make sure the gel is submerged.

Prepare the transfer stack according to the manufacturer's instructions.

Transfer proteins using BioRad pre-programmed protocols: Mixed MW setting (2.5A for 0h 7m 0s).

Transfer PVDF membrane into 5 % milk/TBS-T and block for 1h 0m 0s at RT with constant shaking.

Prepare primary antibodies in 5 % milk/TBS-T and incubate membranes in antibody solution at 4°C with constant shaking

A	B	C
Target	Catalogue no (Supplier)	Dilution
pan-phospho-Rab	ab230260 (Abcam)	1:1000
Rab8A	6975S (Cell Signaling)	1:1000
Rab10 pT73	ab230261 (Abcam)	1:1000
Rab10	8127S (Cell Signaling)	1:1000
LRRK2 pS935	ab133450 (Abcam)	1:1000
LRRK2	ab133474 (Abcam)	1:1000
beta-Actin	A1978-200UL (Sigma-Aldrich)	1:5000

Table 1: Antibodies which are commonly used in our lab. Note: multiplexing for phopsho- and total protein signal is possible depending on the antibodies used. We strip and re-plot for total protein after detection of the phospho signal as all LRRK2 and Rab antibodies listed here are raised in rabbit.

Wash membranes by shaking in TBS-T for 5-10 min

Repeat the wash step 2x

Repeat washing steps as above.

Detect signal using appropriate detection equipment.

Depending on the primary antibodies and the detection method used, it might be necessary to strip the blot using a Western Blot Stripping buffer (eg #T7135A, Takara Bio) for 0h 10m 0s at RT and repeat the primary and secondary antibody incubation steps.