Western blot protocol for detecting ATP10B in mouse/rat brain

Veerle Baekelandt, María Sanchiz Calvo, Eduard Bentea

Published: 2021-09-28 DOI: 10.17504/protocols.io.byhfpt3n

Abstract

Protocol for detection of ATP10B in rat and mouse brain tissue by Western blotting

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

Steps

Day 1

Prepare samples for Western blotting :

30µg proteins in 12µL of volume (adjust with milliQ water)
2.4µL of 6X SDS buffer + 10% of β-mercaptoethanol

Vortex samples and spin down

Boil the samples for 0h 10m 0s at 98°C

Load samples, together with 7µL 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.

Run the gel at 80V for 0h 10m 0s

Run the gel at 150V for another 0h 45m 0s

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: 0h 30m 0s, up to 1.0 A, 25 V.

Block membranes for 1h 0m 0s using 5% milk dissolved in PBS-T 0,1% at Room temperature

Incubate membranes 0h 30m 0s in ATP10B HPA034574 (Sigma) diluted 1/500 in 5% milk PBS-T 0,1% at 4°C

Day 2

10.

Wash membranes with PBS-T 0,1% for 10 minutes, 5 times at Room temperature :

PBS-T 0h 10m 0s
PBS-T 0h 10m 0s
PBS-T 0h 10m 0s
PBS-T 0h 10m 0s
PBS-T 0h 10m 0s

11.

Incubate membranes for 2h 0m 0s in secondary antibody solution Goat anti-rabbit HRP (Dako) diluted 1/10000 in 5% milk PBS-T 0,1% at Room temperature

12.