Sep 28, 2021
Western blot protocol for detecting ATP10B in mouse/rat brain
- María Sanchiz Calvo1,
- Eduard Bentea1,
- Veerle Baekelandt1
- 1Katholieke Universiteit Leuven
Protocol Citation: María Sanchiz Calvo, Eduard Bentea, Veerle Baekelandt 2021. Western blot protocol for detecting ATP10B in mouse/rat brain. protocols.io https://dx.doi.org/10.17504/protocols.io.byhfpt3n
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: September 24, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 53511
Keywords: ATP10B, western blot, mouse, rat, brain, ASAPCRN
Abstract
Protocol for detection of ATP10B in rat and mouse brain tissue by Western blotting
Materials
Recipe for 100 mL 1x RIPA buffer :
o 5 mL Tris pH 7.4
o 1 mL Triton x100
o 1 g Deoxycholate
o 3 mL 5M NaCl
o 200 µL 0.5M EDTA (pH 8.0)
o 1 mL 10% SDS
Add water until 100 mL (MilliQ water)
Add protease and phosphatase inhibitors (Pi + PPi) right before use
Reagents used during Western blotting :
o 6X SDS buffer
o PageRuler™ Plus Prestained Protein Ladder
o 3-8% NuPAGE™ Tris-Acetate gel
o NuPAGE™ Tris-Acetate SDS Running Buffer
o Trans-Blot® TurboTM PVDF Membrane
o 5% milk powder dissolved in PBS-T 0.1% (PBS + 0.1% Triton-X100)
o ATP10B HPA034574 (Sigma) primary antibody
o Goat anti-rabbit HRP secondary antibody (Dako)
o ECL Prime chemiluminescence kit (GE Healthcare)
Recipe for 50 mL 6x SDS buffer (160 mM TrisHCl pH 6.8, 2% SDS, 200 mM DTT, 40% glycerol, bromophenol blue):
o 5 mL 1.6M TrisHCl pH 6.8
o 1 g SDS
o 1.54 g DTT
Add water until 25 mL (MilliQ water) and dissolve
o 20 mL glycerol
Add water until 50 mL (MilliQ water)
Add spatula tip of bromophenol blue
Before start
Perform protein extraction from snap-frozen brain tissue:
- weigh tissue
- add RIPA buffer (see Materials) : 10 X of the weight (40 mg = 400 µL)
- homogenize samples using sample homogenizer
- sonicate samples at 4 degrees C, 3 times 15 seconds (keep the samples on ice between each sonication)
- centrifuge samples at 6000 g for 10 minutes at 4 degrees C
- collect supernatant and measure protein concentration
- aliquot protein extracts and store at -20 degrees C
Day 1
Day 1
4h 5m
4h 5m
Prepare samples for Western blotting :
- 30 µg proteins in 12 µL of volume (adjust with milliQ water)
- 2.4 µL of 6X SDS buffer + 10% of β-mercaptoethanol
30m
Vortex samples and spin down
Boil the samples for 00:10:00 at 98 °C
10m
Load samples, together with 7 µL of mass marker (PageRuler™ Plus Prestained Protein Ladder) on a 3-8% NuPAGE™ Tris-Acetate gel. Use NuPAGE™ Tris-Acetate SDS Running Buffer for migration.
30m
Run the gel at 80V for 00:10:00
10m
Run the gel at 150V for another 00:45:00
45m
Transfer the proteins on PVDF membrane (Trans-Blot® TurboTM PVDF Membrane) using Trans-Blot Turbo Transfer System (Bio-Rad), using the pre-programmed protocol STANDARD SD: 00:30:00 , up to 1.0 A, 25 V.
30m
Block membranes for 01:00:00 using 5% milk dissolved in PBS-T 0,1% at Room temperature
1h
Incubate membranes Overnight in ATP10B HPA034574 (Sigma) diluted 1/500 in 5% milk PBS-T 0,1% at 4 °C
30m
Day 2
Day 2
3h 10m
3h 10m
Wash membranes with PBS-T 0,1% for 10 minutes, 5 times at Room temperature :
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
50m
Incubate membranes for 02:00:00 in secondary antibody solution Goat anti-rabbit HRP (Dako) diluted 1/10000 in 5% milk PBS-T 0,1% at Room temperature
2h
Wash membranes with PBS-T 0,1% for 10 minutes, 5 times at Room temperature :
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
- PBS-T 00:10:00
50m
Develop membranes using ECL Prime (GE Healthcare) for at least 00:10:00
10m