TITLE: Quantitative immunoblot analysis of LRRK1 signalling pathway
AUTHORS: Asad  Malik, Athanasios Karapetsas, Francesca Tonelli  , Dario R 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 LRRK1 signalling pathway components in cell and mouse tissue extracts. Specifically, we immunoblot using rigorously validated and characterized antibodies for LRRK1-total, Rab7A and pS72 Rab7A, pT202/Y204 ERK1/2, phosphor(Ser)-PKC Substrates and GAPDH or Tubulin (loading controls), 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. 

[GUIDELINES]
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.

 


[STEPS]
SECTION: Preparation of lysates from cultured cells
1. 
 
Quickly rinse cells in the tissue culture dish by carefully pouring 4 Room temperature culture media without Foetal bovine serum (FBS) into the dish.

SECTION: Preparation of lysates from cultured cells
2. 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 of lysates from cultured cells
3. Immediately transfer the cell dishes to ice.
SECTION: Preparation of lysates from cultured cells
4. 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 from cultured cells
5. Using a pipette, transfer cell lysate to an Eppendorf tube on ice. 
 


SECTION: Preparation of lysates from cultured cells
6. Leave samples on ice for 20 min to allow for efficient lysis.

SECTION: Preparation of lysates from cultured cells
7. Clarify lysates by centrifugation at 20800 x g, 10 min, 4 °C.

SECTION: Preparation of lysates from cultured cells
8. Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes on ice.
 


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

SECTION: Preparation of samples for immunoblot analysis
10. Prepare samples for immunoblotting to achieve the same protein concentration for all samples (ideally, 0.5 µg/µL to 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
11. Incubate samples for 5 min at 70 °C heating block before immunoblot analysis.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
12. 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)
13. 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)
14. 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)
15. 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)
16. Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.
SECTION: Protein transfer (Wet electroblotting)
17. Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for 90 min on ice using a wet transfer system.
SECTION: Protein transfer (Wet electroblotting)
18. 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)
19. Each membrane can be divided into three sections by two horizontal cuts (one cut just above the 75 kDa ladder band and another cut between the 37.5 kDa and 25 kDa ladder bands) (Figure 1): 
‘top section’ (from the top of the membrane to the 75 kDa marker) - to be probed for LRRK1
‘middle section’ (between the 75 kDa and the 37.5 kDa marker) - to be probed for GAPDH/tubulin (loading control)
‘bottom section’ (from the 37.5 kDa marker to the bottom of the membrane) - to be probed for pS72-Rab7A/Rab7A

  

 

SECTION: Membrane blocking and antibody incubation
20. Destain membranes from Step 19 by washing with TBS-T and incubate in blocking solution for at least 15 min at 70 Room temperature on a see-saw rocker.

SECTION: Membrane blocking and antibody incubation
21. 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:
 Table 1:
      ­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    pT202/Y204 ERK1/2  Cell Signalling Technology  #9101  Rabbit  1 mg/ml    Phospho-(Ser) PKC Substrate Antibody  Cell Signalling Technology  #2261  Rabbit  1 mg/ml    LRRK1 (total) (C-terminus)  MRC-PPU Reagents and Services,   University of Dundee  S405C  Sheep  1 mg/ml    GAPDH  Antibodies Inc./NeuroMab  sc-32233  Mouse  1:5,000    alpha-tubulin  Cell Signaling Technology  3873  Mouse  1:5,000  
 
SECTION: Membrane blocking and antibody incubation
22. After incubation with primary antibodies, wash membranes in TBS-T on a see-saw rocker (3 washes, 5-10 minutes per wash).
SECTION: Membrane blocking and antibody incubation
23. 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.
 

Table 2: 
      Secondary Antibodies  Company  Cat. number  Notes    goat   anti-mouse IRDye 680LT  LI-COR   926-68020       goat   anti-mouse IRDye 800CW   LI-COR   926-32210       goat   anti-rabbit IRDye 800CW   LI-COR   926-32211       donkey   anti-mouse IRDye 680LT  LI-COR   926-68022       donkey   anti-mouse IRDye 800CW   LI-COR   926-32212       donkey   anti-rabbit IRDye 800CW   LI-COR   926-32213       donkey   anti-goat IRDye 800CW  LI-COR   926-32214  Reacts with Sheep primary Abs  
 
SECTION: Membrane blocking and antibody incubation
24. Extensively wash membranes in TBS-T on a see-saw rocker (4 washes, 10-15 minutes per wash).
SECTION: Image acquisition and Analysis
25. 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
26. Analyse immunoblotting data using a software for statistical analysis (Graphpad Prism, or equivalent). 



  

Figure 2. Representative results of quantitative immunoblotting analysis of pSer72 Rab7A/total Rab7A and total LRRK1 levels performed in extracts from wild-type (WT) and homozygous LRRK1 knock-out (KO) mouse embryonic fibroblasts treated +/- PKC activator PMA (100 ng/ml, 30 min) according to the protocol described here.