Quantitative immunoblot analysis of LRRK1 signalling pathway

Francesca Tonelli, Dario Alessi, Asad Malik, Athanasios Karapetsas

Published: 2022-06-02 DOI: 10.17504/protocols.io.6qpvr68e3vmk/v1

Abstract

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.

Steps

Preparation of lysates from cultured cells

Note

Note : To ensure LRRK1 activation in cells, we recommend treating cells for at least 0h 30m 0s ± PMA (a PKC activator) at a final concentration of 100ng/mL (or equivalent volume of DMSO) before lysis.

Quickly rinse cells in the tissue culture dish by carefully pouring 4Room temperature culture media without Foetal bovine serum (FBS) into the dish.

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.

Note

Note : The amount of lysis buffer to use will depend on cell type and cell confluency. As a guideline, use 0.1mL to 0.2mL of lysis buffer for each well of a 6-well plate, 0.5mL for a 10- cm dish and 1mL for a 15-cm dish. It is preferable to aim for protein concentrations of a least 0.75 as this will enable the appropriate amount of protein to be loaded onto a gel as detailed below.

Immediately transfer the cell dishes to ice.

Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.

Using a pipette, transfer cell lysate to an Eppendorf tube On ice.

Note

Note : For non-adherent cells, transfer cells to a Falcon tube and pellet by centrifugation at 180x g,°C,0h 3m 0s; wash cells once with 4Room temperature culture media without FBS 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.

Leave samples On ice for 0h 20m 0s to allow for efficient lysis.

Clarify lysates by centrifugation at 20800x g,4°C.

Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes On ice.

Note

Note : Cell lysates can be snap frozen in liquid nitrogen and stored at -80°C for future use.

Preparation of samples for immunoblot analysis

Determine the protein concentration of cell or tissue lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.

Note

Note: 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 mg/ml (depending on cell type), while tissues lysed as described above should result in protein concentrations of at least 2 mg/ml.

10.

Prepare samples for immunoblotting to achieve the same protein concentration for all samples (ideally, 0.5 to 2, 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 7.5µL of lysate/lysis buffer mix, add 2.5µL of loading buffer). Mix by vortexing.

11.

Incubate samples for 0h 5m 0s at 70°C heating block before immunoblot analysis.

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.

Note

Notes : The amount of protein loaded for each sample ranges from 10µg to 40µg, depending on the cell/tissue 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 10µg to 15µg of protein for each cell extract for optimal signal; for mouse tissues like brain, spleen, kidney, lung and intestine we recommend loading 30µg to 40µg 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.

13.

Electrophorese samples at 130V with MOPS SDS running buffer for 2h 0m 0s or until the blue dye runs off the gel.

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.

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.

Note

Note : Carefully remove any air bubbles between layers using a roller after adding each layer.

16.

Carefully close the cassette holder and insert it in the transfer tank. Fill the tank with transfer buffer.

17.

Electrophoretically transfer proteins from gel onto a nitrocellulose membrane at 100 V (constant voltage) for 1h 30m 0s 70On ice using a wet transfer system.

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.

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

Note

Notes :When immunoblotting using anti-phospho(Ser)-PKC Substrates, it is preferable to blot the entire membrane to visualise all induced PKC substrates.The same blot can be simultaneously be blotted with a loading control like alpha tubulin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pT202/Y204 ERK1/2 (to confirm that stimulation of cells with PMA has worked).

Membrane blocking and antibody incubation

20.

Destain membranes from Step 19 by washing with TBS-T and incubate in blocking solution for at least 0h 15m 0s at 70Room temperature on a see-saw rocker.

21.

Rinse the membrane in TBS-T and incubate 0h 15m 0s 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:

A	B	C	D	E
Antibody Target	Company	Cat. number	Host species	Dilution
pS72 Rab7A	Abcam Inc.	MJF-38, Clone 1	Rabbit	1 ug/ml
Rab7A (Total)	Sigma	R8779	Mouse	1 ug/ml
pT202/Y204 ERK1/2	Cell Signalling Technology	#9101	Rabbit	1 ug/ml
Phospho-(Ser) PKC Substrate Antibody	Cell Signalling Technology	#2261	Rabbit	1 ug/ml
LRRK1 (total) (C-terminus)	MRC-PPU Reagents and Services, University of Dundee	S405C	Sheep	1 ug/ml
GAPDH	Antibodies Inc./NeuroMab	sc-32233	Mouse	1:5,000
alpha-tubulin	Cell Signaling Technology	3873	Mouse	1:5,000

22.

After incubation with primary antibodies, wash membranes in TBS-T on a see-saw rocker (3 washes, 5-10 minutes per wash).

23.

Incubate membranes with near-infrared fluorescent dye-labelled secondary antibodies (diluted to the working concentration: 1:20,000) for 1h 0m 0s at 4Room temperature on a see-saw rocker.

Note

Note :If multiplexing primary antibodies, use secondary antibodies labelled with spectrally distinct near-infrared 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.

Table 2:

A	B	C	D
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

24.

Extensively wash membranes in TBS-T on a see-saw rocker (4 washes, 10-15 minutes per wash).

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.

Note

Note : 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.

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.

Quantitative immunoblot analysis of LRRK1 signalling pathway

Abstract

Steps

Preparation of lysates from cultured cells

Preparation of samples for immunoblot analysis

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Protein transfer (Wet electroblotting)

Membrane blocking and antibody incubation

Image acquisition and Analysis

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