Jul 25, 2022

Public workspaceWestern blotting for LRRK2 signalling in macrophages

DOI
dx.doi.org/10.17504/protocols.io.3byl4k67jvo5/v1
  • 1Department of Comparative Biomedical Science, Royal Veterinary College, Royal College Street, London, NW1 0TU, U.K;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
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DOI: dx.doi.org/10.17504/protocols.io.3byl4k67jvo5/v1
Protocol Citation: sherbst 2022. Western blotting for LRRK2 signalling in macrophages. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4k67jvo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 14, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 50741
Keywords: Western Blot, LRRK2, Rab GTPase, phosporylation, NuPAGE, Turbo Blot, ASAPCRN
Funders Acknowledgement:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Abstract
This protocol describes the immunoblotting for components of the LRRK2 signalling pathway (LRRK2, LRRK2 pS935 and phospho-Rabs) using Invitrogen NuPage SDS-PAGE reagents and the BioRad Turbo Blot transfer system.
Materials
Reagents:

  • Lysis buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% (v/v) Triton X-100
NOTE: add protease and phosphatase inhibitors fresh each time (eg Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), #78440, ThermoFisher Scientific)
  • Loading buffer: NuPAGE LDS sample buffer (#NP0007, ThermoFisher Scientific)
  • Sample reducing agent: NuPAGE sample reducing agent (#NP0009, ThermoFisher Scientific)
  • 4-12% Bis-Tris NuPAGE gels (eg #NP0321BOX, ThermoFisher Scientific)
  • SDS-PAGE running buffer: MES running buffer (#NP0002, ThermoFisher Scientific)
  • Trans-Blot® Turbo™ PVDF Transfer Packs: eg #1704157, BioRad
  • TBS-T: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20.
  • Blocking and antibody dilution buffer: 5 % (w/v) non-fat milk powder in TBS-T
  • Primary and secondary antibodies (see table 1 for primary antibody suggestions)

Equipment:

  • Gel tank (#A25977, ThermoFisher Scientific; or similar)
  • Trans-Blot® Turbo™ Transfer System (1704150, BioRad)
  • + standard lab equipment such as a shaker, bench top centrifuge and heat block
Preparation of protein lysates
Preparation of protein lysates
Culture cells as usual in a 12-well or 6-well plate.
Note
We recommend including a control using an LRRK2 kinase inhibitor (eg MLi-2, 100 nM for 1 hr) in your sample preparation to verify phospho-Rab immunoblots.

Wash cells once in PBS
Wash
Add 100 μl per 12-well or 200 μl per 6 well of lysis buffer containing protease and phosphatase inhibitors. Keep samples TemperatureOn ice from now on.

Scrape cells using a cell lifter and transfer lysate to a 1.5 ml Eppendorf tube
Vortex and incubate on ice for Duration00:10:00 .

10m
Clarify lysate by spinning at Centrifigation17000 x g at Temperature4 °C for Duration00:15:00

15m
Transfer supernatant into a fresh 1.5 ml Eppendorf tube and store at -20 °C.
Note
  1. Determine protein concentration using an assay of choice (eg Pierce™ Coomassie Plus Assay Kit)

SDS-PAGE
SDS-PAGE
Prepare protein lysates by adding sample loading buffer and reducing agent (eg NuPAGE LDS sample buffer and reducing agent).
Note
The final protein concentration should be in the range of Concentration1-2 µg/µL . We recommend loading Amount10-15 µg of total protein per lane.


Denature samples at Temperature80 °C for Duration00:08:00

8m
Prepare the SDS gel (4-12% Bis-Tris Nu-PAGE gel) by assembling the apparatus, filling the tank with MES running buffer and flushing all the wells with MES running buffer.
Spin down the samples quickly in case of condensation and load wells carefully. Include a broad range molecular weight marker (eg Broad molecular weight ladder, ab116028, Abcam).
Run the gel at 180 V for Duration00:35:00 .

35m
Transfer
Transfer
Transfer SDS gel into 20% EtOH while preparing the transfer stack. Make sure the gel is submerged.
Prepare the transfer stack according to the manufacturer's instructions.
Transfer proteins using BioRad pre-programmed protocols: Mixed MW setting (2.5A for Duration00:07:00 ).

7m
Immunolabelling and detection
Immunolabelling and detection
Transfer PVDF membrane into 5 % milk/TBS-T and block for Duration01:00:00 at RT with constant shaking.

Note
We routinely cut the membrane into three parts to be able to blot for LRRK2, a loading control and Rabs at the same time.

1h
Prepare primary antibodies in 5 % milk/TBS-T and incubate membranes in antibody solution at Temperature4 °C DurationOvernight with constant shaking


ABC
TargetCatalogue no (Supplier)Dilution
pan-phospho-Rabab230260 (Abcam)1:1000
Rab8A6975S (Cell Signaling)1:1000
Rab10 pT73ab230261 (Abcam)1:1000
Rab108127S (Cell Signaling)1:1000
LRRK2 pS935ab133450 (Abcam)1:1000
LRRK2 ab133474 (Abcam)1:1000
beta-ActinA1978-200UL (Sigma-Aldrich)1:5000
Table 1: Antibodies which are commonly used in our lab. Note: multiplexing for phopsho- and total protein signal is possible depending on the antibodies used. We strip and re-plot for total protein after detection of the phospho signal as all LRRK2 and Rab antibodies listed here are raised in rabbit.


Wash membranes by shaking in TBS-T for 5-10 min

Repeat the wash step 2x
Wash
Repeat washing steps as above.
Wash
Detect signal using appropriate detection equipment.
Optional: stripping and re-blot
Optional: stripping and re-blot
10m
10m
Depending on the primary antibodies and the detection method used, it might be necessary to strip the blot using a Western Blot Stripping buffer (eg #T7135A, Takara Bio) for Duration00:10:00 at RT and repeat the primary and secondary antibody incubation steps.
10m