Apr 15, 2026

Auto-(de)phosphorylation assay

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxw3rwv8j/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxw3rwv8j/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Auto-(de)phosphorylation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxw3rwv8j/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: December 09, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 114608
Keywords: ASAPCRN, P5B-type ATPase, ATP13A4, Auto-phosphorylation assay, p5b atpases like atp13a4, atp13a4 phospho, atp13a4, p5b atpase, phosphorylation, type atpase, phosphorylated intermediate, induced dephosphorylation, phosphate, enzyme level, assay
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes an assay to measure ATP13A4 phospho-enzyme levels. ATP13A4, a P-type ATPase, forms a phosphorylated intermediate on a conserved aspartate residue during its catalytic cycle. In P5B ATPases like ATP13A4, spontaneous auto-phosphorylation occurs, followed by polyamine-induced dephosphorylation. The assay utilizes SDS-PAGE under acidic conditions, preserving the stability of the aspartyl-phosphate intermediate, followed by autoradiography to detect [32P]-labeled phosphorylated intermediates.

Materials
Buffers and solutions

Stacking gel buffer
  • 516 mM Tris-HCl (pH 5.8) 
 
Running gel buffer
  • 516 mM Tris-HCl (pH 6.8)
 
10x EP buffer
  • 1.6 M KCl 
  • 170 mM Hepes 
  • 20 mM MgCl2
  • 50 mM NaN3
  • 10 mM DTT (add before use)
 
Stop Solution
  • 20% trichloroacetic acid 
  • 10 mM phosphoric acid 
 
Sample buffer
  • 5 mM NaH2PO4, pH 6.0 (NaOH)
  • 0.005% SDS
  • 0.1 g/ml LDS
  • 10% glycerol
  • 0.5 mg/ml bromophenol blue
  • 20 µl/ml β-mercaptoethanol (add before use)
 
Running buffer  
  • 170 mM MOPS, pH 6.3 (Tris) 
  • 0.1% SDS
 

Reagents

  • 30% acrylamide/bis-acrylamide solution, 37.5:1 (Cat# 1610158; Bio-Rad)
  • APS: Ammonium persulfate (Cat# A3678; Sigma-Aldrich )
  • TEMED: N,N,N′,N′-Tetramethylethylenediamine (Cat# T9281; Sigma-Aldrich)
  • DTT: Dithiothreitol (Cat# A2948.0025; VWR)
  • Tris: Trizma base (Cat# T1503; Sigma-Aldrich)
  • MOPS: γ-(N-Morpholino)propanesulphonic acid (Cat# A1076.1000, VWR)
  • KCl: Potassium chloride (Cat# P9541; Sigma-Aldrich)
  • Hepes (Cat# H3375; Sigma-Aldrich)
  • MgCl2.6H2O: Magnesium chloride hexahydrate (Cat# M2670; Sigma-Aldrich)
  • NaN3: Sodium azide (Cat# 1.06688.0100; VWR)
  • Trichloroacetic Acid (Cat# T9159; Sigma-Aldrich)
  • Sodium dodecyl sulfate (SDS) (Cat# 71725; Sigma-Aldrich)
  • Phosphoric acid (Cat# 424045000; Thermo Fisher)
  • Hydroxylamine (Cat# 438227-50ML; Sigma-Aldrich)
  • Sodium phosphate monobasic (NaH2PO4) (Cat# S0751; Sigma-Aldrich)
  • LDS: Lithium dodecyl sulfate (Cat# L4632; Sigma-Aldrich)
  • Glycerol (Cat# 3783.5; Carl Roth)
  • Bromophenol blue (Cat# 17132901, PlusOne)
  • β-mercaptoethanol (Cat# M3148; Sigma-Aldrich)
  • Acetic acid (Cat# 27225; Sigma-Aldrich)
  • Radioactive ATP: ATP, [γ-32P]- 3000Ci/mmol 10mCi/ml EasyTide, 500 µCi (Cat# BLU502A100UC, Revvity, Inc.)
  • GE Storage Phosphor Screens (Cat# GE28-9564-75; Merck)
Safety warnings
The assay must be conducted in a designated radioactive area by authorized personnel, adhering strictly to all radioactive material handling precautions. Appropriate personal protective equipment, including gloves, lab goggles, lab coats, and a dosimeter for monitoring exposure, must be worn at all times. Work should be carried out behind Plexiglass screens, and only filter-equipped pipette tips should be used. Institutional regulations must be followed for the storage of radioactive materials and the disposal of both solid and liquid radioactive waste.

Preparation of acidic gels
Prepare mixes for running gels and stacking gels:
ComponentsRunning gelStacking gel
30% acrylamide/bis-acrylamide10 ml1.6 ml
H2024.6 ml8.52 ml
*TEMED30 ul19.2 ul
*APS 10%300 ul200 ul
SDS 10%400 ul120 ul
Running gel buffer5 mlN/A
Stacking gel bufferN/A1.52 ml
* Add these components right before pouring the gel!

Pour running gel in the glass plates assembled with spacers.
Add butanol on top of the running gel and wait until the running gel is solidified to wash the butanol off with water.
Pour stacking gel on top and insert a suitable comb.
Once the stacking gel is solidified, store at 4 °C .

Running assay
5h 40m
Add membranes (40 µg protein) or purified protein (1 µg ), 10 µL 10x EP buffer solution, milliQ water and whatever you need (compound to test) in a final volume of 95 µL .
Note
Keep samples on ice!


To start the reaction: add 5 µL radioactive ATP mix. Vortex.

To prepare radioactive ATP mix (2 μCi, 5.125 micromolar (µM) ), pipet together 5 µL [γ-32P] ATP + 110.5 µL 120 micromolar (µM) cold ATP

For phosphorylation only: stop reaction with 400 µL stop solution after desired time.

For dephosphorylation: 30 s after addition of [γ-32P] ATP, add compound to test (e.g. non-radioactive ATP, ADP and/or polyamines) and stop reaction with 400 µL stop solution after desired time.
Leave samples on ice for 00:30:00 to precipitate protein.

30m
20.000 x g, 4°C for 00:30:00 .

30m
Aspirate supernatant with pipet, keep in disposable recipient.
Wash pellet with stop solution (400 µL ), and 20.000 x g, 4°C for 00:10:00 .

10m
Repeat step 12 and 13.
Wash pellet with 0.3 M hydroxylamine (HA) (200 µL ), incubate for 00:15:00 and 20.000 x g, 4°C for 00:10:00 .

25m
Dissolve pellet in 40 µL sample buffer.

Load 40 µL on acidic gels.

Note
Very important to load gel carefully, avoid overflow between wells!


Run the gel at 100 V, 04:00:00 4 °C .

4h
When the gel run is complete, carefully cut off the blue dye front. This is excess 32P running ahead of the samples and is not related to ATP13A4 phosphoenzyme which runs at 130 kDa.
Soak gel for 00:05:00 in 7.5% acetic acid, and put gel in plastic bag.

5m
Expose gel Overnight to phosphoimager cassette.

Image cassette using a phosphor imager (e.g. Typhoon‱ FLA 7000, GE Healthcare).
To verify equal sample loading, soak the gel in Coomassie stain Overnight , then wash and evaluate the staining intensity.
Protocol references
van Veen S, Martin S, Van den Haute C, Benoy V, Lyons J, Vanhoutte R, et al. ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 2020;578(7795):419-24.

Sorensen DM, Holemans T, van Veen S, Martin S, Arslan T, Haagendahl IW, et al. Parkinson disease related ATP13A2 evolved early in animal evolution. PLoS One. 2018;13(3):e0193228.