TITLE: LRRK1 Immunoprecipitation kinase assay
AUTHORS: Asad  Malik, Athanasios Karapetsas, Dario R Alessi

[DESCRIPTION]
We describe a non-radioactive assay that we deploy for analysing LRRK1 protein kinase activity in vitro using Rab7A as a substrate. This assay can be used to measure the intrinsic activity of LRRK1 immunoprecipitated from cells as well as to assess the impact of mutations on LRRK1 activity.

[STEPS]
SECTION: Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
1. Transfect HEK293 cells at around 60-70% confluency. For a 10cm dish, add 10 µg DNA (GFP-tagged LRRK1 or GFP-empty vector) and 30 µL of 1 mg/mL PEI solution to 1 mL of Opti-MEMTM Reduced Serum Medium and vortex for 20/30 seconds.
 






SECTION: Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
2. Incubate at Room temperature for 20 min to allow the DNA/PEI complexes to form.

SECTION: Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
3. Add the transfection mix to the culture medium in each dish and incubate cells at 37 °C after transfection.

SECTION: Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
4. Lyse cells 20-24 hours after transfection.
SECTION: Preparation and quantification of cell lysates:
5. Quickly rinse cells in the tissue culture dish by carefully pouring Room temperature culture media without Foetal bovine serum (FBS) into the dish.

SECTION: Preparation and quantification of cell lysates:
6. 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.
 

SECTION: Preparation and quantification of cell lysates:
7. Immediately transfer the cell dishes to ice.
SECTION: Preparation and quantification of cell lysates:
8. Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.
SECTION: Preparation and quantification of cell lysates:
9. Using a pipette, transfer cell lysate to an Eppendorf tube on ice. 

SECTION: Preparation and quantification of cell lysates:
10. Leave samples on ice for 20 min to allow for efficient lysis.

SECTION: Preparation and quantification of cell lysates:
11. Clarify lysates by centrifugation at 20800 x g, 10 min, 4 °C. 
SECTION: Preparation and quantification of cell lysates:
12. Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes on ice.
 


SECTION: Preparation and quantification of cell lysates:
13. Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate. 
 

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
14. 
 
Add 20 mL of aGFP16-aGFP2-His6 NHS-activated Sepharose beads (washed 3 times in PBS and resuspended in PBS to make a 1:1 slurry) to 1 mg of cell extract.
 
     
SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
15. Incubate at 4 °C for 180 min, under mild agitation.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
16. Collect the resin by centrifugation at 2500 x g, 2 min, 4 °C. Discard supernatant.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
17. Resuspend resin in 500 µL of lysis buffer supplemented with 300 millimolar (mM) NaCl.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
18. Repeat steps 16 and 17 twice.
SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
19. Collect the resin by centrifugation at 2500 x g, 2 min, 4 °C. Discard supernatant.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
20. Resuspend resin in 500 µL of 50 millimolar (mM) HEPES pH 7.5.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
21. Repeat step 16 and 20.
SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
22. Collect the resin by centrifugation at 2500 x g, 2 min, 4 °C. Discard supernatant.

SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
23. Resuspend the resin in 50 millimolar (mM) HEPES pH 7.5 (1:1 ratio).
SECTION: Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
24. Aliquot the resin into Eppendorf tubes kept on ice (one Eppendorf tube per reaction, 10 µL resin each).

SECTION: In vitro kinase assay:
25. 
 
Prepare a “master mix” containing 50 millimolar (mM) HEPES pH 7.5, 50 millimolar (mM) KCl, 10 millimolar (mM) MgCl2, 1 millimolar (mM) ATP, and recombinant Rab protein.

SECTION: In vitro kinase assay:
26. 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, 1000 rpm.

SECTION: In vitro kinase assay:
27. Stop the kinase reaction by adding 4X LDS loading buffer to the reaction mix to a final concentration of 2X.
SECTION: In vitro kinase assay:
28. Incubate the mixture at 70 °C on a heat block for 10 min to elute LRRK1 from the resin.

SECTION: In vitro kinase assay:
29. Collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.
SECTION: In vitro kinase assay:
30. Supplement the samples with 2‐Mercaptoethanol to 1% (v/v).
SECTION: In vitro kinase assay:
31. Incubate the samples for 5 min at 70 °C on a heat block before proceeding to quantitative immunoblotting analysis.

SECTION: Analysis of kinase reaction products by quantitative immunoblotting analysis
32. The reaction products can be analysed by quantitative immunoblotting analysis (as described in XXXX). Table 1 lists the primary antibodies that we recommend using, which include antibodies to detect Rab7A phosphorylation at Serine-72.
 
      ­Antibody   Target  Company  Cat. number  Host species  Dilution    pS72 Rab7A  Abcam Inc.  MJF-38, Clone 1  Rabbit  1 mg/ml    Rab7A (Total)  Sigma  R8779  Mouse  1 mg/ml    LRRK1 (total) (C-terminus)  MRC-PPU Reagents and Services,   University of Dundee  S405C  Sheep  1 mg/ml    alpha-tubulin  Cell Signaling Technology  3873  Mouse  1:5,000