Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

1. Sample Preparation for the P-tag SDS–PAGE

1. Remove and discard the media from 10 cm dishes in which cells are grown using a suction and wash cells with 5 mL Dulbecco's phosphate-buffered saline (DPBS) by first adding DPBS to the side of the dishes to avoid disturbing the cell layer, and manually rock the dishes back and forth several times.
2. Remove and discard the DPBS using a suction and add 2 mL of 0.25% (w/v) trypsin diluted in DPBS, and gently rock the dishes to cover the cell layer. Put the dishes into a CO₂ incubator (37 °C, humidified air, 5% CO₂) for 5 min.
3. After pipetting up and down using a disposable pipette to break up detached cells, collect the cell suspension into a 15 mL tube and measure the cell density using a hematocytometer under a microscope¹⁸.
4. Dilute the cells to 2.5 × 10⁵ cells/mL with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Add 2 mL (5 × 10⁵ cells) of the diluted cell suspension to each well of 6-well plates.
5. Grow the cells overnight in a CO₂ incubator (37 °C, humidified air, 5% CO₂).
6. Transfection into HEK293 cells
   NOTE: If phosphorylation of endogenous Rab10 is to be examined, proceed to step 1.7. Plasmids used in this protocol can be obtained from the authors upon request. See Table of Materials for brief information and Figure S1 for their DNA sequences.
   1. Aliquot 200 µL of DMEM into 1.5 mL tubes.
   2. To each tube, add both 0.266 µg (final concentration of 1.33 µg/mL) of a plasmid encoding HA-Rab10 and 1.066 µg (final concentration of 5.33 µg/mL) of a plasmid encoding 3×FLAG-LRRK2 from 500 µg/mL plasmid stocks. Then add 4 µL of 1 mg/mL solution of polyethyleneimine (dissolved in 20 mM HEPES-NaOH, pH 7.0) and immediately mix the solution by vortexing for 5 s.
   3. Let the tubes stand at room temperature for 10 min and add the content of one tube dropwise to one well using a micropipette. Gently and manually rock the culture plates back and forth several times to let the transfection mixture diffuse evenly throughout the well.
7. Let the cells grow for another 24-36 h in a CO₂ incubator (37 °C, humidified air, 5% CO₂).
8. If phosphorylation of endogenous Rab10 is to be examined, treat cells with and without LRRK2 inhibitors for 1 h before lysing cells.
   1. Prepare stock solutions of LRRK2 inhibitors by dissolving the inhibitors in dimethyl sulfoxide (DMSO) at 10 mM. We recommend using MLi-2 and GSK2578215A, which are highly specific and potent LRRK2 inhibitors. Store stock solutions at -80 °C.
   2. Prepare working stocks of the inhibitors by diluting the stock solutions with DMSO: For MLi-2, prepare 10 µM and 30 µM for endogenous and overexpressed LRRK2, respectively. For GSK2578215A, prepare 1 mM and 3 mM for endogenous and overexpressed LRRK2, respectively.
   3. Add 2 µL of the working stocks of either MLi-2 or GSK2578215A to the middle of one well using a micropipette and gently rock the plate manually back and forth several times to let the inhibitors diffuse evenly throughout the well.
   4. Add 2 µL of DMSO to a different well as a negative control in a similar manner to the inhibitor in step 1.8.3.
   5. Put the plates back to the incubator and culture the cells for 1 h.
9. Lyse the cells.
   1. Put the culture plates on ice. Remove and discard the media. Wash the cells by first adding 2 mL DPBS to the side of the dishes to avoid disturbing the cell layer and manually rock the dishes back and forth several times.
   2. Remove and discard the DPBS, and add to the cells 100 µL of the lysis buffer (50 mM Tris-HCl pH 7.5, 1% (v/v) polyoxyethylene(10) octylphenyl ether, 1 mM EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid), 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 µg/mL microcystin-LR, 270 mM sucrose, protease inhibitor cocktail).
   3. Tilt the plates on ice and scrape the cells using a cell scraper to gather as much cell lysate as possible. Collect the lysates using a micropipette into 1.5 mL tubes (pre-chilled on ice).
      Caution: Microcystin-LR can be fatal if swallowed or in contact with skin.
10. Let the tubes stand on ice for 10 min for complete lysis. Clarify the cell lysates by centrifugation (20,000 × g, 10 min at 4 °C) and transfer the supernatants to new 1.5 mL tubes pre-chilled on ice.
11. Measure protein concentration (µg/µL) of the cleared lysates by Bradford assay.
    1. Prepare bovine serum albumin (BSA) standards (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) by diluting the stock solution with distilled water. Dilute the cleared cell lysates by 20-fold with distilled water.
    2. Put 5 µL/well of the BSA standards, blank (distilled water), and each diluted cell lysate into a 96-well plate in triplicate.
    3. Add 150 µL/well of the Bradford assay reagent using 12-channel micropipette and let the plate stand at room temperature for 5 min.
    4. Measure the absorbance at 595 nm on a plate reader and compare to BSA standards.
12. Prepare 100 µL of samples for SDS-PAGE. The protein concentration of the samples is 1 µg/µL for overexpressed HA-Rab10 and 2 µg/µL for endogenous Rab10.
    1. Using the quantified concentrations from step 1.11.4, calculate the volume (µL) of the cell lysates equivalent to 100 µg (overexpressed HA-Rab10) or 200 µg (endogenous Rab10) by dividing the protein amount (100 µg or 200 µg) by the protein concentration of lysates (µg/µL).
    2. Add 25 µL of 4× Laemmli's SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 4% (v/v) β-mercaptoethanol) to new 1.5 mL tubes kept at room temperature.
    3. Add the calculated volume of the cell lysates to each tube and mix by vortexing for 5 s at room temperature.
    4. Bring the total volume to 100 µL with the lysis buffer and mix by vortexing for 5 s at room temperature.
13. Supplement with 10 mM MnCl₂ to quench the chelating agent. Add 1 µL of 500 mM MnCl₂. Mix by vortexing for 5 s at room temperature.
    NOTE: Samples containing MnCl₂ may not be suitable for normal SDS-PAGE. This step is required due to the presence of the chelating agent (such as ethylenediaminetetraacetic acid (EDTA), EGTA, etc.) in the lysis buffer. Otherwise, Mn²⁺ ions coupled to P-tag acrylamide will be dissociated by the chelating agents, and P-tag SDS-PAGE will not work properly.
14. Boil all samples at 100 °C for 5 min and store the samples below -20 °C until use. The boiled samples can be stored up to at least 6 months.

2. Casting Gels for P-tag SDS–PAGE

NOTE: Gels should be made on the same day as running the gels. Gels can be made under ambient light conditions.

1. Prepare 5 mM P-tag acrylamide stock solution by first dissolving the 10 mg of powder/solid P-tag acrylamide completely with 100 µL of methanol and then bring to 3.3 mL by adding double distilled water.
   NOTE: P-tag acrylamide is light sensitive. The prepared solution should be stored in the dark at 4 °C until use.
2. Clean both plain and notched plates by spraying 70% ethanol and wiping with a paper towel. Assemble the gel plates. The dimensions of the gel plates used in this particular protocol are 80 or 100 mm long by 100 mm wide. Clean silicon spacers are put in between plain and notched plates and the assembled plates are clamped with binder clips.
   NOTE: Any type of gel plates that work for ordinary SDS-PAGE can be used.
3. Put a comb to be used (17-well plastic comb) into the assembled gel plates and mark on the gel plate the position of the bottom of the wells with a permanent marker.
4. Prepare 10 mL of 10% acrylamide gel mixture (10% (w/v) acrylamide (acrylamide:bis-acrylamide = 29:1), 375 mM Tris-HCl (pH 8.8), 0.1% (w/v) SDS) in a 15 mL tube.
   NOTE: The optimal concentration of acrylamide may vary depending on the reagents used. Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) should not be added at this point.
5. Add 100 µL of 5 mM P-tag acrylamide and 10 µL of 1 M MnCl₂ solutions at final concentrations of 50 µM and 100 µM, respectively.
   NOTE: The optimal concentrations of the P-tag acrylamide and MnCl₂ might also vary depending on the reagents used.
6. Add 15 µL of TEMED to the gel mixture at a final concentration of 0.15% (v/v) and then 50 µL of 10% (w/v) APS at a final concentration of 0.05% (w/v). Mix well by gently swirling the tube for 5 s and pour into the assembled plates immediately up to a height that is 2 mm below the position marked in step 2.3.
7. Gently layer 2-propanol onto the gel solution to flatten the top of separating gels.
8. Let the gels stand for 30 min at room temperature. It is not necessary to protect the gels from light.
   NOTE: It might take longer for gels to set under cold room temperature. Degassing the gel mixture before adding TEMED and APS helps accelerate this step. For this purpose, the gel mixture can be prepared in a 100-200 mL Erlenmeyer flask connected to a suction. Degas the solution for 10 min.
9. Remove the layered 2-propanol by absorbing with a paper towel.
10. Wash the top space of the gels by filling the space up with distilled water from a wash bottle and discard the water by pouring off into basin. Repeat washing 3 times.
11. Remove residual water remaining in the top space of the gels by absorbing with a paper towel.
12. Prepare 3 mL of 4% acrylamide gel mixture (4% (w/v) acrylamide (a: crylamide:bis-acrylamide = 29:1), 125 mM Tris-HCl (pH 6.8), 0.1% (w/v) SDS) in a 15 mL tube.
    NOTE: Do not add P-tag acrylamide or MnCl₂ solution to the stacking gel mixture.
13. Add 7.5 µL of TEMED and 24 µL of 10% (w/v) APS at final concentrations of 0.25% (v/v) and 0.08% (w/v), respectively. Mix well by gently swirling the tube for 5 s and pour the mixture on top of the separation gel. Immediately put appropriate combs (17-well plastic combs which accommodate up to 25 µL of samples, for example).
14. Let the gels stand for 30 min at room temperature. It is not necessary to protect the gels from light. After the stacking gels set, run them without further storage.

3. SDS–PAGE and Immunoblotting

1. Remove the combs from the gels. Then remove the silicon spacers and then clips from the gels.
2. Put the casted gels into gel tanks and fix the gel to the tank by clamping with the binder clips.
3. Pour the running buffer (25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS) at the bottom and top of the gels. Clean wells by flushing the running buffer using a 5 mL syringe and a 21G needle to remove gel pieces.
4. Remove air bubbles from the bottom space of the gels using a bent needle attached to a syringe. To make a bent needle, manually bend a 21G needle in the middle of the needle so that the angle between the tip and the base of the needle becomes 30-45 °.
5. Spin down precipitates caused by the addition of MnCl₂ at 20,000 × g for 1 min at room temperature to obtain clear samples.
6. Load 10 µg proteins for detecting the phosphorylation of overexpressed Rab10 and 30 µg proteins for endogenous Rab10.
   NOTE: It is critical to load equal volume of samples in ALL wells. Empty lanes should be loaded with 1× Laemmli's SDS-PAGE sample buffer. If the samples contain MnCl₂, add the same concentration of MnCl₂ to the dummy samples.
7. The well loaded with a molecular weight marker (MWM) should also be supplemented with 1× Laemmli's SDS-PAGE sample buffer so the volume of samples loaded is the same in all wells.
   NOTE: Again, if the samples contain MnCl₂, add the same concentration of MnCl₂ to the MWM. Alternatively, an EDTA-free MWM can be used.
8. Run gels at 50 V for stacking (approximately 30 min) until the dye front crosses into the separation gel.
9. After the samples stack, change the voltage to 120 V for separation until the dye front reaches the bottom of the gels (approximately 50 and 80 min for 80 and 100 mm long gels, respectively).
   NOTE: The migration pattern of the MWM is expected to be different from that on normal SDS-PAGE gels. It should not be used for estimating the molecular weight of proteins on P-tag SDS-PAGE gels but can be used for checking the reproducibility of P-tag gels. Refer to Discussion for details.
10. Wash the gels to remove MnCl₂ from the gels.
    1. Pour the transfer buffer (48 mM Tris, 39 mM Glycine, 20% (v/v) methanol) containing 10 mM EDTA and 0.05% (w/v) SDS into a container (e.g., large weighing boats). The volume of the buffer should be sufficient to cover a gel.
    2. Remove the separation gels from the gel plates and put one gel in one container. Discard the stacking gel.
    3. Leave the gels on a rocking shaker (~60 rpm) for 10 min at room temperature.
    4. Repeat the wash steps in total 3 times.
       NOTE: Use fresh buffer for each wash.
    5. Wash the gels once for 10 min with the transfer buffer containing 0.05% (w/v) SDS to remove EDTA. The volume of the buffer should be sufficient to cover a gel.
11. Electro-transfer to nitrocellulose or polyvinylidene difluoride (PVDF) membranes using wet tanks.
    1. Place a filter paper (10 x 7 cm) on a sponge pad for transfer. Place the gel on the filter paper. Make sure there are no air bubbles between the filter paper and the gel.
    2. Put a membrane (10 x 7 cm) on the gel and make sure there are no air bubbles between the gel and the membrane.
    3. Put another filter paper (10 x 7 cm) on the membrane and, again, make sure there are no air bubbles between the membrane and the filter paper.
    4. Put another sponge pad on the filter paper. Put the stack of membrane/filter papers in a cassette for transfer.
    5. Put the cassette into a transfer tank, making sure that the membrane is located between the gel and the positively charged anode.
    6. Connect the tank to a power supply and put the tank in a styrene foam box filled with ice-cold water. Start transfer at 100 V for 180 min.
       NOTE: Prolonged transfer is necessary since the transfer of proteins from P-tag SDS-PAGE gels are not as efficient as that from normal SDS-PAGE gels. Efficient cooling is essential to avoid melting gels during transfer.
12. Check the transfer
    1. Remove the membranes from the gels using tweezers and soak the membranes in a Ponceau S solution (0.1% (w/v) Ponceau S, 5% (v/v) acetic acid) to stain transferred proteins on the membranes in a plastic container. The volume of the solution should be sufficient to cover a membrane.
    2. Incubate the membranes for 1 min at room temperature by rocking manually.
       NOTE: A ladder of bands should become visible in each lane.
    3. Pick the membrane up out of the staining solution with tweezers and see if the ladder of bands has uniform pattern and intensity in every lane where the samples have been loaded.
    4. After checking the staining, remove the staining solution and add TBST buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% (v/v) polyoxyethylenesorbitan monolaurate). The volume of the buffer should be sufficient to cover a membrane.
       NOTE: The Ponceau S solution can be collected and re-used several times.
    5. Rock the membranes in TBST on a rocking shaker (~60 rpm) at room temperature until no visible bands remain on the membranes.
    6. Repeat the washing step with fresh TBST for 5 min.
13. Remove and discard the TBST. Block by adding 5% (w/v) skim milk dissolved in TBST and rocking on a shaker (~60 rpm) for 1 h at room temperature. The volume of the blocking solution should be sufficient to cover a membrane.
14. Prepare primary antibody solutions by diluting primary antibodies (anti-Rab10 antibody for endogenous Rab10 and anti-HA antibody for overexpressed HA-Rab10) in 10 mL per membrane of the blocking solution (see Table of Materials for concentrations).
15. Remove and discard the blocking solution and add the primary antibody. Incubate the membranes on a rocking shaker (~60 rpm) overnight at 4 °C.
16. Remove the primary antibody solution and add TBST to wash the membranes. The volume of the buffer should be sufficient to cover a membrane.
17. Incubate the membranes for 5 min on a rocking shaker (~60 rpm) at room temperature. Repeat the wash (step 3.16) in total 3 times using fresh TBST each time.
18. Prepare secondary antibody solutions by diluting secondary antibody labeled with horseradish peroxidase (HRP) in 10 mL per membrane of the blocking solution. Use anti-rabbit IgG antibody labeled with HRP for membranes probed with the anti-Rab10 antibody, and anti-mouse IgG antibody labeled with HRP for those probed with the anti-HA antibody (see Table of Materials for concentration).
19. Remove and discard the TBST after the third wash and add the secondary antibody solution. Incubate the membranes on a rocking shaker (~60 rpm) for 1 h at room temperature.
20. Wash the membranes in TBST for 10 min. Repeat the wash step in total 3 times, similar to step 3.17.
21. Develop the membranes using enhanced chemiluminescence (ECL).
    NOTE: Exposure time might vary depending on the ECL solution and the system used for detection of chemiluminescence.
    1. Turn on an imager equipped with a charge-coupled device (CCD) camera and a computer connected to the imager. Start up the control software for the imager. Wait until the temperature of the CCD camera has reached -25 °C.
    2. Put 1 mL of the ECL solution for one membrane on a plastic wrap spread on a bench.
    3. Put a membrane gel-side-up on the ECL solution and then quickly flip it over so that both sides of the membrane are coated with the solution.
    4. Pick the membrane up and drain it by letting one side of the membrane touch a paper towel for 5 s.
    5. Put the membrane between clear films (e.g., clear paper pockets).
       NOTE: Plastic wrap is not recommended for wrapping the membranes. The clear films used for wrapping the membrane need to be as flat as possible without visible wrinkles to avoid uneven background.
    6. Place the membrane on a black tray. Put the tray in the imager and close the door.
    7. Click the "Focusing" button in the window of the control software. Check that the membrane is correctly positioned. Click the "Return" button.
    8. Select "Precision" for "Exposure Type". Select "Manual" for "Exposure Time" and set the exposure time to 1 s.
    9. Select "High" for "Sensitivity/Resolution". Leave "Add Digitization Image" unchecked. Click the "Start" button to take an image.
    10. Save the image that appeared on the display in the computer as a TIFF file.
    11. Repeat taking images with the exposure time from 1, 3, 10, 30, 60, 90, 120, 150 s and up to 180 s. When taking the last image, check "Add Digitization Image" so the digital image, not the chemiluminescence, of the membrane can be simultaneously taken.
    12. Select the best image with the largest dynamic range and without any saturating pixels (shown in red) in the bands of interest.
        NOTE: Conventional X-ray films can also be used for detection¹⁹.