Jul 09, 2024

Public workspaceWestern blotting to detect ATP13A2

 Forked from Western blotting to detect ATP13A2 and ATP13A3
DOI
dx.doi.org/10.17504/protocols.io.e6nvwdqz2lmk/v1
  • 1Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase, MD 20815, USA
Stephanie Vrijsen
Open access
DOI: dx.doi.org/10.17504/protocols.io.e6nvwdqz2lmk/v1
Protocol Citation: Stephanie Vrijsen, Marine Houdou, Peter Vangheluwe 2024. Western blotting to detect ATP13A2. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdqz2lmk/v1
Manuscript citation:

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: December 05, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91838
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
Protocol to detect ATP13A2 via Western Blotting.
Materials

  • Antibodies:

- Goat anti-mouse IgG (H+L) secondary antibody HRP conjugated: Thermo Scientific, 31430
- Goat anti-rabbit IgG (H+L) secondary antibody HRP conjugated: Thermo Scientific, 31460
- Mouse monoclonal anti-GAPDH antibody (lot #067M4785V, dilution 1:5,000): Sigma, G8795.
- Rabbit anti-ATP13A2 antibody (lot #0000102992, dilution 1:1,000): Sigma, A3361.

  • TrypLE: Thermo Scientific, 11528856

  • Dulbecco’s Phosphate Buffered saline modified without calcium chloride and magnesium chloride (DPBS): Gibco, D8537

  • Micro-BCA Protein Assay Kit: Pierce BCA Protein Assay Kit, Thermo Scientific, 23225

  • NuPAGE LDS sample buffer: Invitrogen, NP0007

  • Pre-cast 4-12% Bis-Tris gels: Invitrogen, NP0321BOX

  • PVDF membranes: Thermo Scientific, 88518

  • RIPA Lysis and Extraction Buffer: Invitrogen: 89900

  • SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free: Sigma: S8830

  • SuperSignal West Pico PLUS chemiluminescent Substrate: Thermo Scientific, 34095

  • Transfer buffer: Life Technologies, NP0006-1

  • MES buffer: Thermo Scientific, NP0002
Harvesting cells
Harvesting cells
Collect the cells by using TrypLE.
Centrifuge cell suspensions at 450 xg, 4°C for Duration00:05:00 .

5m
Resuspend cell pellets with DPBS and centrifuge following the same indications as in 2. Repeat once.
Discard supernatants and keep cell pellets on ice.
Cell lysis and protein concentration determination
Cell lysis and protein concentration determination
Resuspend cell pellets in RIPA buffer (RIPA Lysis and Extraction Buffer) supplemented with protease cocktail inhibitors.
Vortex Duration00:00:30 and keep on ice for Duration00:10:00 .

10m 30s
Centrifuge at 20,000g, 4 °C for Duration00:10:00 .

10m
Keep supernatants on ice to proceed with protein concentration determination using the micro-BCA Protein Assay Kit.
SDS-PAGE and Western blot
SDS-PAGE and Western blot
7h 10m
Loading
Mix 10 µg of protein with NuPAGE LDS sample buffer and 5% β-mercaptoethanol final.
Boil for Duration00:05:00 at 95 °C

5m
Load protein on pre-cast 4-12% Bis-Tris gels. Include at least one lane with a protein ladder.
Running
Run for and Duration01:20:00 at 130V in MES buffer.

1h 20m
Transfer
Transfer onto PVDF membranes using a liquid transfer and following settings: 100V, Duration01:30:00 , 4°C. Use transfer buffer with 10% methanol.

1h 30m
Blocking
Duration01:00:00 Block membranes with blocking buffer (5% milk powder in 1X TBS and 0.1% Tween20 (REF)) for Duration01:00:00 at room temperature, 19 rpm.

2h
Primary antibodies
Incubate membrane with primary antibodies in solution (1% bovine serum albumin in 1X TBS-Tween20 (TBS-T) buffer), DurationOvernight at 4°C, 19 rpm.

1h
Wash membrane three times for Duration00:05:00 in TBS-T, 19 rpm.

5m
Secondary antibodies
Incubate membrane with peroxidase-conjugated secondary antibodies in solution (1% milk powder in 1X TBS-T) for Duration01:00:00 at room temperature and, 19 rpm.

1h
Wash membrane five times forDuration00:05:00 in TBS-T, 19 rpm.

5m
Detection
Use a chemiluminescence reagent to detect signal and acquire with a Biorad Camera (Vilber Lourmat) and its software (ImageLab).