Assessment of in vitro kinase activity of over-expressed and endogenous LRRK2 immunoprecipitated from cells

Transfect HEK293 cells at around 60-70% confluency. For a 10cm dish, add 10µg DNA (FLAG-tagged LRRK2 or FLAG empty vector) and 30µL of 1mg/mL PEI solution to 1mL of Opti-MEM^TM Reduced Serum Medium and vortex for 20/30 seconds.

Incubate at Room temperature for 0h 20m 0s to allow the DNA/PEI complexes to form.

Add the transfection mix to the culture medium in each dish drop by drop using a pipette and incubate cells at 37°C after transfection.

Lyse cells 20h 0m 0s -24h 0m 0s after transfection.

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

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.

Immediately transfer the cell dishes to ice.

Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) 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 0h 20m 0s to allow for efficient lysis.

Clarify lysates by centrifugation at 20800x g,0h 0m 0s for 0h 10m 0s at 4°C.

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

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

Add 20µL of ANTI-FLAG® M2 Affinity Gel (washed 3 times in PBS and resuspended in PBS to make a 1:1 slurry so that 20 µl of 1:1 slurry correspond to 10 µl resin) to 1mg of cell extract.

Incubate at 4°C for 1h 0m 0s, under mild agitation.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend resin in 500µL of lysis buffer supplemented with 300millimolar (mM) NaCl.

Repeat step 16 and 17 twice.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend resin in 500µL of 50millimolar (mM) Tris-HCl 7.5.

Repeat step 16 and 20.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend the resin in 50millimolar (mM) Tris-HCl 7.5 (1:1 ratio, i.e. resuspend 10 µl resin in 10 µl Tris-HCl).

Aliquot the resin into Eppendorf tubes kept On ice (one Eppendorf tube per reaction, 10µL resin each).

For each reaction, couple 10µg of anti-LRRK2 antibody (UDD3, available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/) to 20µL Protein A/G Sepharose resin (washed 3 times with PBS and resuspended in PBS to make a 1:1 slurry so that 20 µl of 1:1 slurry correspond to 10 µl resin) by incubating for 1h 0m 0s at 4°C under mild agitation. Include a control where UDD3 is replaced by pre-immune IgG.

To get rid of any unbound antibody, wash the antibody/resin complexes: collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C, discard supernatant and resuspend in 250µL of PBS.

Repeat step 26 twice more.

Add the antibody/resin mix to 5mg of cell extract per reaction.

Incubate at 4°C for 1h 0m 0s, under mild agitation.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend resin in 250µL of lysis buffer supplemented with 300millimolar (mM) NaCl.

Repeat step 30 and 31 twice more.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend resin in 250µL of 50millimolar (mM) Tris-HCl 7.5.

Repeat step 33 and 34.

Collect the resin by centrifugation at 2500x g,0h 0m 0s for 0h 2m 0s at 4°C. Discard supernatant.

Resuspend the resin in 50millimolar (mM) Tris-HCl 7.5 (1:1 ratio).

Aliquot the resin into Eppendorf tubes kept On ice (one Eppendorf tube per reaction, 20µL resin each).

Prepare a “master mix” containing 50millimolar (mM) Tris-HCl 7.5, 10millimolar (mM) MgCl2, 1millimolar (mM) ATP, and recombinant Rab protein.

Start the kinase reaction by adding the master mix to the immunoprecipitated kinase and transferring the Eppendorf tubes to the thermo mixer set at 30°C, 1000rpm,0h 0m 0s.

Stop the kinase reaction by adding 4X LDS loading buffer to the reaction mix to a final concentration of 2X.

Incubate the mixture at 70°C on a heat block for 0h 10m 0s to elute LRRK2 from the resin.

Collect the eluent by centrifugation through a 0.22μm-pore-size Spinex column.

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

Incubate the samples for 0h 5m 0s at 70°C on a heat block before proceeding to quantitative immunoblotting analysis.

Once the in vitro kinase assay has been performed, we recommend analysing the reaction products by quantitative immunoblotting (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6).

Figure 2: In vitro phosphorylation of recombinant Rab10 by LRRK2 immunoprecipitated from mouse embryonic fibroblasts (MEFs). In vitro phosphorylation of recombinant Rab10 by LRRK2 immunoprecipitated from mouse embryonic fibroblasts (MEFs). Endogenous LRRK2 was immunoprecipitated from 4 mg cell extracts derived from littermate matched wild-type and LRRK2^-/-(knock-out), or littermate matched wild-type and LRRK2^[G2019S]KI MEFs. The immunoprecipitates were subjected to an in vitro kinase reaction at 30°C in the presence of 1 mg recombinant Rab10 and excess Mg-ATP, with or without 1µM LRRK2 inhibitor MLi-2. The reactions were terminated after 1 hour and analyzed by immunoblotting with the indicated antibodies. The membranes were developed using the Odyssey CLx scan Western Blot imaging system.