TITLE: Co-immunoprecipitation protocol to study LRRK2 binding to Rab12 in a cell-based assay
AUTHORS: Francesca Tonelli

[DESCRIPTION]
This protocol describes the immunoprecipitation (IP) of FLAG-tagged LRRK2 from whole cell lysates to assess its interaction with Rab12. This method can be used to screen the impact that LRRK2 mutations have on its binding to Rab12 in cells, as well as the effect of any compound or cell treatment on LRRK2 interaction with Rab12.

Note: The IP products can be analysed by quantitative immunoblotting (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6) using an antibody targeting Rab12.

[GUIDELINES]
This protocol describes the immunoprecipitation (IP) of FLAG-tagged LRRK2 from whole cell lysates to assess its interaction with Rab12. This method can be adapted to immunoprecipitate HA-tagged Rab12 from whole cell lysates to assess its interaction with LRRK2 by replacing ANTI-FLAG® M2 Affinity Gel with an anti-HA antibody-coupled matrix (Pierce™ Anti-HA Agarose, ThermoFisher Scientific, Cat #26181, or equivalent).

[STEPS]
SECTION: Transient transfection of HEK293 cells:
1. Seed HEK293 cells to be 70-90% confluent at transfection. Proceed to the next step the day after seeding cells.
SECTION: Transient transfection of HEK293 cells:
2. Dilute FLAG-tagged LRRK2 (wild-type or mutant) DNA and HA-tagged Rab12 (or HA-empty control) DNA in Opti-MEMTMReduced 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-MEMTMReduced 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.
SECTION: Transient transfection of HEK293 cells:
3. Dilute Lipofectamine®2000 Reagent in Opti-MEMTM 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-MEMTM Reduced Serum Medium.
SECTION: Transient transfection of HEK293 cells:
4. Add the diluted DNA to the diluted Lipofectamine®2000 Reagent and mix gently.
SECTION: Transient transfection of HEK293 cells:
5. Incubate at room temperature for 5 minutes.
SECTION: Transient transfection of HEK293 cells:
6. Add the transfection mix to the culture medium in each dish drop by drop using a pipette.
SECTION: Transient transfection of HEK293 cells:
7. Transfer the plates to a humidified incubator maintaining 37°C and 5% (v/v) CO2.
SECTION: Transient transfection of HEK293 cells:
8. Lyse cells 20-24 hours after transfection, as detailed in the next section.
SECTION: Preparation and quantification of cell lysates:
9. 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.
SECTION: Preparation and quantification of cell lysates:
10. 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. 
SECTION: Preparation and quantification of cell lysates:
11. Immediately transfer the cell dishes to ice.
SECTION: Preparation and quantification of cell lysates:
12. Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.
SECTION: Preparation and quantification of cell lysates:
13. Using a pipette, transfer cell lysate to an Eppendorf tube on ice. 
SECTION: Preparation and quantification of cell lysates:
14. Leave samples on ice for 20 minutes to allow for efficient lysis. 
Note: Mix the samples by inverting the tubes. Do not vortex.
SECTION: Preparation and quantification of cell lysates:
15. Clarify lysates by centrifugation at 15,000 g for 10 min at 4°C. 
SECTION: Preparation and quantification of cell lysates:
16. Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the lysates on ice.
SECTION: Preparation and quantification of cell lysates:
17. 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.
SECTION: Preparation and quantification of cell lysates:
18. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
19. Wash the required amount of ANTI-FLAG® M2 Affinity Gel beads (25 µl of packed gel beads are required for each immunoprecipitation reaction): 
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
19.1. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
19.2. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
19.3. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
20. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
21. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
22. Collect the resin by centrifugation at 1,500 g for 3 minutes at 4oC. Discard supernatant (flow-through).
Note: The flow-through may be retained if desired to assess efficient depletion of FLAG-LRRK2 from cell lysates.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
23. 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).
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
24. Repeat steps 22 and 23 for a total of 3 washes.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
25. Collect the resin by centrifugation at 1,500 g for 3 minutes at 4oC. Discard supernatant.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
26. 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.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
27. Collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spin-X column.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
28. Supplement the samples with 2‐Mercaptoethanol to 1% (v/v). 
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
29. Incubate the samples for 5 min at 70°C on a heat block.
SECTION: Immunoprecipitation of FLAG-tagged LRRK2 from cell lysates:
30. Proceed to quantitative immunoblotting analysis of the IP products, as detailed in the next section.
SECTION: Analysis of immunoprecipitation products by quantitative immunoblotting analysis:
31. 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.