Co-immunoprecipitation protocol to study LRRK2 binding to Rab12 in a cell-based assay

Seed HEK293 cells to be 70-90% confluent at transfection. Proceed to the next step the day after seeding cells.

Dilute FLAG-tagged LRRK2 (wild-type or mutant) DNA and HA-tagged Rab12 (or HA-empty control) DNA in Opti-MEM^TMReduced Serum Medium in a sterile Eppendorf tube.

For a 10 cm dish : Dilute 8 µg of FLAG-tagged LRRK2 (wild-type or mutant) DNA and 2 µg of HA-tagged Rab12 (or HA-empty control) DNA in 0.5 mL Opti-MEM^TMReduced Serum Medium.

Note: The method described here will also enable to assess the interaction of FLAG-LRRK2 with endogenous Rab12, independent of HA-Rab12 overexpression.

Dilute Lipofectamine®2000 Reagent in Opti-MEM^TM Reduced Serum Medium in another sterile Eppendorf tube.

For a 10 cm dish : Dilute 30 µl of Lipofectamine®2000 Reagent in 0.5 mL of Opti-MEM^TM Reduced Serum Medium.

Add the diluted DNA to the diluted Lipofectamine®2000 Reagent and mix gently.

Incubate at room temperature for 5 minutes.

Add the transfection mix to the culture medium in each dish drop by drop using a pipette.

Transfer the plates to a humidified incubator maintaining 37°C and 5% (v/v) CO₂.

Lyse cells 20-24 hours after transfection, as detailed in the next section.

Quickly rinse cells in the tissue culture dish by carefully pouring room temperature culture media without Foetal bovine serum (FBS) into the dish.

Note: As HEK293 cells are loosely attached to the dish surface, extra care should be taken during the washing step.

Pour off media from the culture dish and completely aspirate any residual media. Immediately add freshly prepared ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer.

Note: As a guideline, use 1 ml of lysis buffer for a 10 cm dish for HEK293 cells.

Immediately transfer the cell dishes to ice.

Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.

Using a pipette, transfer cell lysate to an Eppendorf tube on ice.

Leave samples on ice for 20 minutes to allow for efficient lysis.

Note: Mix the samples by inverting the tubes. Do not vortex.

Clarify lysates by centrifugation at 15,000 g for 10 min at 4°C.

Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the lysates on ice.

Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.

Note: Ensure the concentration of the samples is in the linear range for the Bradford assay. If it isn’t, prepare appropriate dilutions in water of each lysate. Generally, protein concentrations of near confluent cells lysed as described above should result in protein concentrations of at least 2 mg/ml.

Proceed to FLAG-LRRK2 immunoprecipitation, as detailed in the next section.

Note:

-We recommend confirming the expression of the transiently expressed proteins (FLAG-LRRK2 and HA-Rab12) by performing quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6.

-When comparing multiple FLAG-tagged variants of LRRK2, we recommend assessing the expression levels of LRRK2 in the whole lysates by immunoblotting prior to immunoprecipitation and adjusting how much cell lysate is to be used to immunoprecipitate LRRK2 accordingly, to ensure that the amount of LRRK2 between samples is as close as possible.

Wash the required amount of ANTI-FLAG® M2 Affinity Gel beads (25 µl of packed gel beads are required for each immunoprecipitation reaction):

Mix the affinity gel beads by vortexing until completely resuspended, and immediately aliquot the required volume into a 1.5 mL microcentrifuge tube on ice.

Note: Cut 1 mm off the end of a pipette tip when dispensing/aliquoting the affinity gel beads.

Add IP wash buffer to the beads, vortex and centrifuge for 3 minutes at 1,500 x g. Remove supernatant, being careful not to disturb the beads. Repeat this step for a total of 3 washes.

Note: Each wash should be performed with a volume of IP wash buffer of at least 20 times the total packed gel volume.

Resuspend the washed beads in IP wash buffer to make a 1:1 slurry (e.g., add 100 µl IP wash buffer to 100 µl packed beads). Leave on ice until use.

Add 25 µl of packed gel beads from Step 19.3 (= 50 µl of a 1:1 slurry) to 1 mg of whole cell extract from Step 16.

Note: The immunoprecipitation conditions (amount of resin and amount of cell extract) might need optimisation.

Incubate at 4°C for one hour on a rotator, under mild agitation (20 rpm).

Note: Ensure the resin and lysate are mixing properly. If necessary, top up with lysis buffer.

Collect the resin by centrifugation at 1,500 g for 3 minutes at 4^oC. Discard supernatant (flow-through).

Note: The flow-through may be retained if desired to assess efficient depletion of FLAG-LRRK2 from cell lysates.

Resuspend the resin in IP wash buffer. Disperse the resin by gently inverting the tubes. Do not vortex.

Note: Each wash should be performed with a volume of IP wash buffer of at least 20 times the total packed gel volume (e.g. 500 µl IP wash buffer per 25 µl beads).

Repeat steps 22 and 23 for a total of 3 washes.

Collect the resin by centrifugation at 1,500 g for 3 minutes at 4^oC. Discard supernatant.

Elute FLAG-LRRK2 from the resin by resuspending the resin in 2X Loading buffer (prepared by diluting 4X Loading buffer 1:1 in milliQ water) and incubating at 70°C on a heat block for 10 min.

Note: To ensure optimal elution, use a minimum of 25 µl of 2X Loading buffer for 25 µl of resin.

Collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spin-X column.

Supplement the samples with 2‐Mercaptoethanol to 1% (v/v).

Incubate the samples for 5 min at 70°C on a heat block.

Proceed to quantitative immunoblotting analysis of the IP products, as detailed in the next section.

Perform quantitative immunoblotting analysis of the IP products following the method described in dx.doi.org/10.17504/protocols.io.bsgrnbv6 (Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway).

Load 95% of each IP sample to detect the interacting protein (HA-Rab12 and endogenous Rab12). Loading 5% of each IP sample should be sufficient to detect the bait (FLAG-LRRK2). Use the following primary antibodies for immunoblotting analysis of the IP products:

• Anti-FLAG Antibody: DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) (Cell Signaling Technology, Cat #14793) (RRID:AB_2572291). Use at 1:1,000 dilution.
• Anti-total Rab12 Antibody: Sheep polyclonal antibody (MRC PPU Reagents and Services, University of Dundee; Cat #SA227) (RRID: AB_2921227). Use at 1 µg/ml dilution.