Western blotting to detect ATP13A2 and ATP13A3

Depending on cell type, collect the cells by scrapping them with a scrapper in Dulbecco’s Phosphate Buffered saline modified without calcium chloride and magnesium chloride (DPBS) (SH-SY5Y) or, using 0.25% Trypsin-EDTA (HMEC-1) for which stop enzymatic reaction by adding culture medium.

Centrifuge cell suspensions at 450 g (SH-SY5Y) or 2500 rpm (HMEC-1), 4°C for 0h 5m 0s.

Resuspend cell pellets with DPBS and centrifuge following the same indications as in 2 . Repeat once.

Discard supernatants and keep cell pellets on ice.

Resuspend cell pellets in RIPA buffer (RIPA Lysis and Extraction Buffer supplemented with protease cocktail inhibitors.

Vortex 0h 0m 30s and keep on ice for 0h 30m 0s.

Centrifuge at 20,000g, 4°C for 0h 30m 0s.

Keep supernatants on ice to proceed with protein concentration determination using the micro-BCA Protein Assay Kit.

Loading

Mix 20 µg of protein with NuPAGE LDS sample buffer and 5% β-mercaptoethanol final.

For this specific protocol to detect ATP13A2 and ATP13A3, do not boil samples.

Load protein on pre-cast 4-12% Bis-Tris gels. Include at least one lane with a protein ladder.

Running

Run for 0h 10m 0sat 100V and 1h 30m 0sat 110-130V.

Transfer

Transfer onto PVDF membranes using a liquid transfer and following settings: 100V, 1h 15m 0s, 4°C.

Ponceau staining

Rinse membrane with distilled water.

Incubate membrane with Ponceau staining for 0h 5m 0s, 19 rpm.

Scan membrane if necessary.

Blocking

1h 0m 0s Block membranes with blocking buffer (5% milk powder in 1X TBS and 0.1% Tween20 (REF)) for 1h 0m 0s at room temperature, 19 rpm.

Primary antibodies

Incubate membrane with primary antibodies in solution (1% bovine serum albumin in 1X TBS-Tween20 (TBS-T) buffer), 1h 0m 0sat 4°C, 19 rpm.

Wash membrane three times for 0h 5m 0s in TBS-T, 19 rpm.

Secondary antibodies

Incubate membrane with peroxidase-conjugated secondary antibodies in solution (1% milk powder in 1X TBS-T) for 1h 0m 0sat room temperature and, 19 rpm.

Wash membrane five times for0h 5m 0sin TBS-T, 19 rpm.

Detection

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