TITLE: Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway
AUTHORS: Francesca Tonelli, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Accurate, quantitative analysis of protein expression and modifications (such as phosphorylation) is critical when studying cell signalling. Here we describe our method for efficient immunoblotting analysis of the LRRK2 signalling pathway components in cell and mouse tissue extracts. Specifically, we immunoblot using rigorously validated and characterized antibodies for LRRK2-total and LRRK2-pSer935 (multiplexed), Rab10-total and Rab10-pThr73 (multiplexed), Rab12-total and Rab12-pSer105 (multiplexed) and GAPDH or tubulin (loading control), although the protocol described here can also be applied to different cell components. Included are procedures for sample preparation from cultured cells and mouse tissue, gel electrophoresis, protein transfer, and antibody incubation. </div><div class = "text-block"><span style = "font-style:italic;">Note:</span><span> If analysing cells isolated from human blood (neutrophils, monocytes or PBMCs), please refer to the specific protocols deposited in Protocols.io on how to isolate these cells. These lysates can then be analysed by immunoblotting as described here ( See Section "Preparation of samples for immunoblot analysis" to Section "Image acquisition and Analysis").</span></div></div>

[STEPS]
?. [Preparation of lysates]
Please choose the relevant method for preparation of lysate: Preparation of lysates from cultured cellsPreparation of lysates from mouse tissues
?. [Preparation of lysates]
Quickly wash cells in the tissue culture dish by carefully pouring  that the cells are currently growing in into the dish. We do not recommend using Phosphate Buffered Saline to wash cells prior to cell lysis as this may cause nutrient stress.
[culture media without Foetal bovine serum (FBS)]
?. [Preparation of lysates]
Pour off media without FBS from the culture dish, tilt dish and completely aspirate all residual media. Immediately add freshly prepared ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer.
The amount of lysis buffer to use will depend on cell type and cell confluency. As a guideline, use 0.1-0.2 ml of lysis buffer for each well of a 6 well plate, 0.5 ml for a 10 cm dish and 1 ml for a 15 cm dish. It is preferable to aim for protein concentrations of a least 0.75 µg/µl as this will enable the appropriate amount of protein to be loaded onto a gel as detailed below.
?. [Preparation of lysates]
Immediately place the cell dishes  .
on ice
?. [Preparation of lysates]
Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.
?. [Preparation of lysates]
Using a pipette, transfer cell lysate to an Eppendorf tube .
on ice
For non-adherent cells, transfer cells to a Falcon tube and pellet by centrifugation at  ; wash cells once with   and pellet again as above. Discard supernatant and add freshly prepared ice-cold lysis buffer. Immediately transfer the Falcon containing the cell pellet to ice.
?. [Preparation of lysates]
Leave samples  for  to allow for efficient lysis.
on ice
?. [Preparation of lysates]
Clarify lysates by centrifugation at .
Centrifuge: 20800 34, 10 min, 4 10
?. [Preparation of lysates]
Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes .
on ice
Cell lysates can be snap frozen in liquid nitrogen and stored at   for future use.
?. [Preparation of samples for immunoblot analysis]
Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.
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 range from 0.5 to 5 µg/µl (depending on cell type).
?. [Preparation of lysates]
To ensure the specificity of the phosphorylation signals detected by immunoblotting in cell lysates, we recommend treating cells for at least 30 min ± a LRRK2 kinase inhibitor such as MLi-2 at 100nM final concentration (or equivalent volume of DMSO vehicle) before lysis.
?. [Preparation of lysates]
To ensure the specificity of the phosphorylation signals detected by immunoblotting in tissue lysates, we recommend administering mice with MLI-2 (30 mg/kg final dose) by subcutaneous injection for 2 hours prior to tissue collection.
?. [Preparation of lysates]
Collect tissues from mice culled by cervical dislocation and snap freeze in liquid nitrogen immediately after harvesting. Store at  until further use.
-80 °C
?. [Preparation of lysates]
Ground frozen tissue in liquid nitrogen to a fine powder using a pulveriser kit.
?. [Preparation of lysates]
Using a spatula, transfer the tissue powder to an Eppendorf tube on dry ice.
Maintain all Pulverizer units in liquid nitrogen including spatula. After each use wash the pulveriser with tap water and clean thoroughly with 70% (v/v) ethanol.
?. [Preparation of lysates]
Weigh tissue powder.
Keep samples on dry ice during weighing.
?. [Preparation of lysates]
Immediately add a 10-fold volume excess of ice-cold lysis buffer to the tissue powder (for example, add 1000 μl of lysis buffer to 100 mg of tissue powder) and quickly vortex to mix.
?. [Preparation of lysates]
Leave samples  for  to allow for efficient lysis.
on ice
?. [Preparation of lysates]
Clarify lysates by centrifugation at .
Centrifuge: 20800 34, 30 min, 4 10
?. [Preparation of lysates]
Transfer the supernatants into new Eppendorf tubes and discard the pellet.
?. [Preparation of samples for immunoblot analysis]
Prepare samples for immunoblotting to achieve the same protein concentration for all samples (ideally, 0.5-2 µg/µl, depending on the sample at the lowest concentration) by combining the cell lysate with lysis buffer. Add a quarter of a volume of 4X SDS/LDS loading buffer freshly supplemented with beta-mercaptoethanol (i.e. for 7.5 µl of lysate/lysis buffer mix, add 2.5 µl of loading buffer). Mix by vortexing.
?. [Preparation of samples for immunoblot analysis]
Incubate samples for  at  heating block before immunoblot analysis.
70 °C
?. [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)]
Load samples onto a NuPAGE 4–12% Bis–Tris Midi Gel (ThermoFisherScientific, Cat#WG1402BOX or Cat#WG1403BOX), or a self-cast 10% Bis-Tris gel, alongside pre-stained molecular weight markers (ranging from 10 kDa to 250 kDa). Rinse wells carefully with running buffer before loading samples.
The amount of protein loaded for each sample ranges from 10 to 40 μg, depending on the cell type and the protein(s) of interest. For cell lines like mouse embryonic fibroblasts, A549 cells and cells isolated from human peripheral blood (monocytes, neutrophils), we recommend loading 10-15 μg of protein for each cell extract for optimal signal.Be aware of maximum loading capacity of each well as per manufacturer’s instructions and take care not to overload wells. If multiple gels are used for each set of experimental samples, an internal loading control should also be included for subsequent data normalization.
?. [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)]
Electrophorese samples at 130V with MOPS SDS running buffer for  or until the blue dye runs off the gel.
?. [Protein transfer (Wet electroblotting)]
Equilibrate the gel, one piece of nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 μm NC) and two pieces of filter paper (WhatmanTM 3MM Chr Chromatography Paper, or equivalent) (all of the same size as the gel) by pre-soaking them in transfer buffer.
?. [Protein transfer (Wet electroblotting)]
Assemble the gel and membrane transfer stack in a tray filled with transfer buffer to ensure that all components are submerged during the assembling. Place one sponge pad inside the cassette holder (on the side that will be facing the cathode). Place one piece of filter paper on top of the sponge pad, followed by the gel, nitrocellulose membrane, another piece of filter paper and another sponge pad.
Carefully remove any air bubbles between layers using a roller after adding each layer.
?. [Protein transfer (Wet electroblotting)]
Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
?. [Protein transfer (Wet electroblotting)]
Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for  using a wet transfer system.
on ice
?. [Protein transfer (Wet electroblotting)]
After transfer, stain membranes with Ponceau solution to assess transfer efficiency and general quality of the samples. If an image is required for record, the Ponceau-stained membraned can be scanned.
?. [Protein transfer (Wet electroblotting)]
Each membrane can be divided into three sections by two horizontal cuts (one cut just above the 75 kDa ladder band and another cut just below the 37.5 kDa ladder band): ‘top section’ (from the top of the membrane to the 75 kDa marker), ‘middle section’ (between the 75 kDa and the 37.5 kDa marker), and ‘bottom section’ (from the 37.5 kDa marker to the bottom of the membrane) (Figure 1), to be incubated with primary antibodies as described in Step 23.
Samples loaded onto one gel can be simultaneously blotted for LRRK2-total and LRRK2-pSer935, Rab10-total and Rab10-pThr73 and a loading control like alpha tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). If in addition Rab12-total and Rab12-pSer105 levels are analysed, duplicate gels are required.
?. [Membrane blocking and antibody incubation]
Destain membranes from Step 21 by washing with TBS-T and incubate in blocking solution for at least  at room temperature on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
Rinse the membrane in TBS-T and incubate  at  with primary antibodies (diluted in 5% (w/v) BSA in TBS-T to their working concentration – Table 1), as follows: ‘top section’: incubate with anti-total and phospho-LRRK2 antibodies; ‘middle section’: incubate with anti-alpha tubulin or GAPDH antibody; ‘bottom section’: incubate with anti-total and phospho-Rab antibodies.
4 °C
If assessing protein phosphorylation, we multiplex the phospho-specific antibody with the total antibody detecting the protein of interest. Table 1 lists the primary antibodies most commonly used in our lab to study LRRK2 signalling and their suggested working dilution. The selectivity and specificity of the antibodies suggested in Table 1 have been extensively validated using appropriate controls, including overexpression models and knock-out cell lines. All antibodies listed in Table 1 react with both human and mouse cells/tissues.
Antibody TargetCompanyCat. numberHost speciesDilutionRab10 pThr73Abcam Inc. ab230261Rabbit1 µg/mlRab10 (total)Nanotools0680-100/Rab10-
605B11Mouse1 µg/mlRab10 (total)Abcam Inc. ab237703Rabbit1 µg/mlRab12 pSer105 (mouse) /
pSer106 (human)Abcam Inc. ab256487Rabbit1 µg/mlRab12 (total)MRC-PPU Reagents and
Services, University of
DundeeSA227Sheep1 µg/mlRab12 (total)Proteintech18843-1-APRabbit1 µg/mlLRRK2 pSer935MRC-PPU Reagents and
Services, University of
DundeeUDD2Rabbit1 µg/mlLRRK2 (total) (C-terminus)Antibodies Inc./NeuroMab75-253Mouse1 µg/mlLRRK2 (total) (N-terminus)MRC-PPU Reagents and
Services, University of
DundeeUDD3Rabbit0.1 µg/mlGAPDHAntibodies Inc./NeuroMabsc-32233Mouse1:5,000alpha-tubulinCell Signaling Technology3873Mouse1:5,000
?. [Membrane blocking and antibody incubation]
After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker):
?. [Membrane blocking and antibody incubation]
(Wash 1/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
(Wash 2/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
(Wash 3/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for  at  on a see-saw rocker.
4 Room temperature
If multiplexing primary antibodies, use secondary antibodies labelled with spectrally distinct nearinfrared fluorescent dyes. Generally, we use IRDye 800CW (800 nm channel) secondary antibodies for the phospho-antibodies multiplexed with IRDye 680LT (680 nm channel) secondary antibodies for the corresponding total antibody. Table 2 lists the near-infrared fluorescent dye-labelled secondary antibodies used in our lab.
Secondary AntibodiesCompanyCat. numberNotesgoat anti-mouse IRDye 680LTLI-COR926-68020goat anti-mouse IRDye 800CW LI-COR926-32210goat anti-rabbit IRDye 800CW LI-COR926-32211donkey anti-mouse IRDye 680LTLI-COR926-68022donkey anti-mouse IRDye 800CWLI-COR926-32212donkey anti-rabbit IRDye 800CWLI-COR926-32213donkey anti-goat IRDye 800CWLI-COR926-32214Reacts with Sheep primary Abs
?. [Membrane blocking and antibody incubation]
Extensively wash membranes in TBS-T (4 washes, 10-15 minutes each, with agitation):
?. [Membrane blocking and antibody incubation]
(Wash 1/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 2/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 3/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 4/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Image acquisition and Analysis]
Protein bands are acquired via near infrared fluorescent detection using the Odyssey CLx Imaging System and the signal intensity quantified using the Image Studio Software.
To control for inter-gel variability, the signal intensity of each band can be normalised against the control sample loaded in each gel of a set of experiments.
?. [Image acquisition and Analysis]
Analyse immunoblotting data using a software for statistical analysis (Graphpad Prism, or equivalent).
?. [Preparation of samples for immunoblot analysis]
Determine the protein concentration of tissue lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.
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, tissues lysed as described above should result in protein concentrations of at least 2 µg/µl.
?. [Preparation of samples for immunoblot analysis]
Prepare samples for immunoblotting to achieve the same protein concentration for all samples (ideally, 0.5-2 µg/µl, depending on the sample at the lowest concentration) by combining the lysate with lysis buffer. Add a quarter of a volume of 4X SDS/LDS loading buffer freshly supplemented with beta-mercaptoethanol (i.e. for 7.5 µl of lysate/lysis buffer mix, add 2.5 µl of loading buffer). Mix by vortexing.
?. [Preparation of samples for immunoblot analysis]
Incubate samples for  at  heating block before immunoblot analysis.
70 °C
?. [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)]
Load samples onto a NuPAGE 4–12% Bis–Tris Midi Gel (ThermoFisherScientific, Cat#WG1402BOX or Cat#WG1403BOX), or a self-cast 10% Bis-Tris gel, alongside pre-stained molecular weight markers (ranging from 10 kDa to 250 kDa). Rinse wells carefully with running buffer before loading samples.
The amount of protein loaded for each sample ranges from 10 to 40 μg, depending on the tissue type and the protein(s) of interest. For mouse tissues like brain, spleen, kidney, lung and intestine we recommend loading 30-40 μg of protein for each tissue extract for optimal signal. Be aware of maximum loading capacity of each well as per manufacturer’s instructions and take care not to overload wells. If multiple gels are used for each set of experimental samples, an internal loading control should also be included for subsequent data normalization.
?. [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)]
Electrophorese samples at 130V with MOPS SDS running buffer for  or until the blue dye runs off the gel.
?. [Protein transfer (Wet electroblotting)]
Equilibrate the gel, one piece of nitrocellulose membrane (GE Healthcare, Amersham Protran Supported 0.45 μm NC) and two pieces of filter paper (WhatmanTM 3MM Chr Chromatography Paper, or equivalent) (all of the same size as the gel) by pre-soaking them in transfer buffer.
?. [Protein transfer (Wet electroblotting)]
Assemble the gel and membrane transfer stack in a tray filled with transfer buffer to ensure that all components are submerged during the assembling. Place one sponge pad inside the cassette holder (on the side that will be facing the cathode). Place one piece of filter paper on top of the sponge pad, followed by the gel, nitrocellulose membrane, another piece of filter paper and another sponge pad.
Carefully remove any air bubbles between layers using a roller after adding each layer.
?. [Protein transfer (Wet electroblotting)]
Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
?. [Protein transfer (Wet electroblotting)]
Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for  using a wet transfer system.
on ice
?. [Protein transfer (Wet electroblotting)]
After transfer, stain membranes with Ponceau solution to assess transfer efficiency and general quality of the samples. If an image is required for record, the Ponceau-stained membraned can be scanned.
?. [Protein transfer (Wet electroblotting)]
Each membrane can be divided into three sections by two horizontal cuts (one cut just above the 75 kDa ladder band and another cut just below the 37.5 kDa ladder band): ‘top section’ (from the top of the membrane to the 75 kDa marker), ‘middle section’ (between the 75 kDa and the 37.5 kDa marker), and ‘bottom section’ (from the 37.5 kDa marker to the bottom of the membrane) (Figure 1), to be incubated with primary antibodies as described in Step 23.
Samples loaded onto one gel can be simultaneously blotted for LRRK2-total and LRRK2-pSer935, Rab10-total and Rab10-pThr73 and a loading control like alpha tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). If in addition Rab12-total and Rab12-pSer105 levels are analysed, duplicate gels are required.
?. [Membrane blocking and antibody incubation]
Destain membranes from Step 21 by washing with TBS-T and incubate in blocking solution for at least  at room temperature on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
Rinse the membrane in TBS-T and incubate  at  with primary antibodies (diluted in 5% (w/v) BSA in TBS-T to their working concentration – Table 1), as follows: ‘top section’: incubate with anti-total and phospho-LRRK2 antibodies; ‘middle section’: incubate with anti-alpha tubulin or GAPDH antibody; ‘bottom section’: incubate with anti-total and phospho-Rab antibodies.
4 °C
If assessing protein phosphorylation, we multiplex the phospho-specific antibody with the total antibody detecting the protein of interest. Table 1 lists the primary antibodies most commonly used in our lab to study LRRK2 signalling and their suggested working dilution. The selectivity and specificity of the antibodies suggested in Table 1 have been extensively validated using appropriate controls, including overexpression models and knock-out cell lines. All antibodies listed in Table 1 react with both human and mouse cells/tissues.
Antibody TargetCompanyCat. numberHost speciesDilutionRab10 pThr73Abcam Inc. ab230261Rabbit1 µg/mlRab10 (total)Nanotools0680-100/Rab10-
605B11Mouse1 µg/mlRab10 (total)Abcam Inc. ab237703Rabbit1 µg/mlRab12 pSer105 (mouse) /
pSer106 (human)Abcam Inc. ab256487Rabbit1 µg/mlRab12 (total)MRC-PPU Reagents and
Services, University of
DundeeSA227Sheep1 µg/mlRab12 (total)Proteintech18843-1-APRabbit1 µg/mlLRRK2 pSer935MRC-PPU Reagents and
Services, University of
DundeeUDD2Rabbit1 µg/mlLRRK2 (total) (C-terminus)Antibodies Inc./NeuroMab75-253Mouse1 µg/mlLRRK2 (total) (N-terminus)MRC-PPU Reagents and
Services, University of
DundeeUDD3Rabbit0.1 µg/mlGAPDHAntibodies Inc./NeuroMabsc-32233Mouse1:5,000alpha-tubulinCell Signaling Technology3873Mouse1:5,000
?. [Membrane blocking and antibody incubation]
After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker):
?. [Membrane blocking and antibody incubation]
(Wash 1/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
(Wash 2/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
(Wash 3/3): Wash membranes in TBS-T -, on a see-saw rocker.
?. [Membrane blocking and antibody incubation]
Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for  at  on a see-saw rocker.
4 Room temperature
If multiplexing primary antibodies, use secondary antibodies labelled with spectrally distinct nearinfrared fluorescent dyes. Generally, we use IRDye 800CW (800 nm channel) secondary antibodies for the phospho-antibodies multiplexed with IRDye 680LT (680 nm channel) secondary antibodies for the corresponding total antibody. Table 2 lists the near-infrared fluorescent dye-labelled secondary antibodies used in our lab.
Secondary AntibodiesCompanyCat. numberNotesgoat anti-mouse IRDye 680LTLI-COR926-68020goat anti-mouse IRDye 800CW LI-COR926-32210goat anti-rabbit IRDye 800CW LI-COR926-32211donkey anti-mouse IRDye 680LTLI-COR926-68022donkey anti-mouse IRDye 800CWLI-COR926-32212donkey anti-rabbit IRDye 800CWLI-COR926-32213donkey anti-goat IRDye 800CWLI-COR926-32214Reacts with Sheep primary Abs
?. [Membrane blocking and antibody incubation]
Extensively wash membranes in TBS-T (4 washes, 10-15 minutes each, with agitation):
?. [Membrane blocking and antibody incubation]
(Wash 1/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 2/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 3/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Membrane blocking and antibody incubation]
(Wash 4/4): Extensively wash membranes in TBS-T -, with agitation.
?. [Image acquisition and Analysis]
Protein bands are acquired via near infrared fluorescent detection using the Odyssey CLx Imaging System and the signal intensity quantified using the Image Studio Software.
To control for inter-gel variability, the signal intensity of each band can be normalised against the control sample loaded in each gel of a set of experiments.
?. [Image acquisition and Analysis]
Analyse immunoblotting data using a software for statistical analysis (Graphpad Prism, or equivalent).