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

[DESCRIPTION]
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. 

Note: 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").

In this new version, the last step contains a supplemental video with extra context and tips, as part of the ASAP Protocol Particulars, featuring conversations with protocol authors

[GUIDELINES]
Figures

 
   


[STEPS]
SECTION: Preparation of lysates
1. Please choose the relevant method for preparation of lysate: 
Preparation of lysates from cultured cells
Preparation of lysates from mouse tissues
SECTION: Preparation of lysates
3. Quickly wash cells in the tissue culture dish by carefully pouring Room temperature 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.
SECTION: Preparation of lysates
4. 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.
 

SECTION: Preparation of lysates
5.  Immediately place the cell dishes  on ice.

SECTION: Preparation of lysates
6. 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 of lysates
7. Using a pipette, transfer cell lysate to an Eppendorf tube on ice. 
 

SECTION: Preparation of lysates
8.  Leave samples on ice for 20 min to allow for efficient lysis.

SECTION: Preparation of lysates
9. Clarify lysates by centrifugation at 20800 x g, 10 min, 4 °C. 

SECTION: Preparation of lysates
10. Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes on ice.
 


SECTION: Preparation of samples for immunoblot analysis
11. Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate. 
 

SECTION: Preparation of lysates
2. 
 

SECTION: Preparation of lysates
2. 
 

SECTION: Preparation of lysates
3. Collect tissues from mice culled by cervical dislocation and snap freeze in liquid nitrogen immediately after harvesting. Store at -80 °C until further use.

SECTION: Preparation of lysates
4. Ground frozen tissue in liquid nitrogen to a fine powder using a pulveriser kit.
SECTION: Preparation of lysates
5. Using a spatula, transfer the tissue powder to an Eppendorf tube on dry ice.
 

SECTION: Preparation of lysates
6. Weigh tissue powder.
 

SECTION: Preparation of lysates
7. 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.
SECTION: Preparation of lysates
8. Leave samples on ice for 20 min to allow for efficient lysis.

SECTION: Preparation of lysates
9. Clarify lysates by centrifugation at 20800 x g, 30 min, 4 °C. 

SECTION: Preparation of lysates
10. Transfer the supernatants into new Eppendorf tubes and discard the pellet.
SECTION: Preparation of samples for immunoblot analysis
12. 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.
SECTION: Preparation of samples for immunoblot analysis
13. Incubate samples for 5 min at 70 °C heating block before immunoblot analysis.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
14. 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.
 

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
15. Electrophorese samples at 130V with MOPS SDS running buffer for 120 min or until the blue dye runs off the gel.

SECTION: Protein transfer (Wet electroblotting)
16. 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.
SECTION: Protein transfer (Wet electroblotting)
17. 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.
 

SECTION: Protein transfer (Wet electroblotting)
18.  Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
SECTION: Protein transfer (Wet electroblotting)
19. Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for 100 min on ice using a wet transfer system.
SECTION: Protein transfer (Wet electroblotting)
20. 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.
SECTION: Protein transfer (Wet electroblotting)
21. 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.
 
  
SECTION: Membrane blocking and antibody incubation
22. Destain membranes from Step 21 by washing with TBS-T and incubate in blocking solution for at least 15 min at room temperature on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
23. Rinse the membrane in TBS-T and incubate 15 min at 4 °C 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.
 


 

SECTION: Membrane blocking and antibody incubation
24. After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker):
SECTION: Membrane blocking and antibody incubation
24.1. (Wash 1/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
24.2. (Wash 2/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
24.3. (Wash 3/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
25. Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for 60 min at 4 Room temperature on a see-saw rocker.
 


 

SECTION: Membrane blocking and antibody incubation
26. Extensively wash membranes in TBS-T (4 washes, 10-15 minutes each, with agitation):
SECTION: Membrane blocking and antibody incubation
26.1. (Wash 1/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.2. (Wash 2/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.3. (Wash 3/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.4. (Wash 4/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Image acquisition and Analysis
27. 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.
 

SECTION: Image acquisition and Analysis
28. Analyse immunoblotting data using a software for statistical analysis (Graphpad Prism, or equivalent).
SECTION: Preparation of samples for immunoblot analysis
11. Determine the protein concentration of tissue lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate. 
 

SECTION: Preparation of samples for immunoblot analysis
12. 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.
SECTION: Preparation of samples for immunoblot analysis
13. Incubate samples for 5 min at 70 °C heating block before immunoblot analysis.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
14. 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.
 

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
15. Electrophorese samples at 130V with MOPS SDS running buffer for 120 min or until the blue dye runs off the gel.

SECTION: Protein transfer (Wet electroblotting)
16. 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.
SECTION: Protein transfer (Wet electroblotting)
17. 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.
 

SECTION: Protein transfer (Wet electroblotting)
18.  Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
SECTION: Protein transfer (Wet electroblotting)
19. Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for 100 min on ice using a wet transfer system.

SECTION: Protein transfer (Wet electroblotting)
20. 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.
SECTION: Protein transfer (Wet electroblotting)
21. 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.
 
  
SECTION: Membrane blocking and antibody incubation
22. Destain membranes from Step 21 by washing with TBS-T and incubate in blocking solution for at least 15 min at room temperature on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
23. Rinse the membrane in TBS-T and incubate 15 min at 4 °C 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.
 


 

SECTION: Membrane blocking and antibody incubation
24. After incubation with primary antibodies, wash membranes in TBS-T (3 washes, 5-10 minutes each, on a see-saw rocker):
SECTION: Membrane blocking and antibody incubation
24.1. (Wash 1/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
24.2. (Wash 2/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
24.3. (Wash 3/3): Wash membranes in TBS-T 5 min-10 min, on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
25. Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for 60 min at 4 Room temperature on a see-saw rocker.
 


 

SECTION: Membrane blocking and antibody incubation
26. Extensively wash membranes in TBS-T (4 washes, 10-15 minutes each, with agitation):
SECTION: Membrane blocking and antibody incubation
26.1. (Wash 1/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.2. (Wash 2/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.3. (Wash 3/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Membrane blocking and antibody incubation
26.4. (Wash 4/4): Extensively wash membranes in TBS-T 10 min-15 min, with agitation.

SECTION: Image acquisition and Analysis
27. 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.
 

SECTION: Image acquisition and Analysis
28. Analyse immunoblotting data using a software for statistical analysis (Graphpad Prism, or equivalent).
SECTION: ASAP Protocol Particulars: context and tips
29.