Apr 15, 2026
Purification of ATP13A4 from HEK293T cells
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Purification of ATP13A4 from HEK293T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9z85vzp/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 10, 2024
Last Modified: April 15, 2026
Protocol Integer ID: 114661
Keywords: ASAPCRN, Membrane protein purification, HEK293T, ATP13A4, purification of atp13a4, overexpressing atp13a4, atp13a4, purification, affinity purification, membrane solubilization, cell lysi, using strep
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes the purification of ATP13A4 from HEK293T cells stably overexpressing ATP13A4. The procedure includes cell lysis, membrane solubilization, and affinity purification using Strep-Tactin‱XT resin.
Materials
Buffers
LIS buffer
- 10 mM Tris-HCl (pH 7.5)
- 0.5 mM MgCl₂
- 1 mM DTT
- SigmaFast protease inhibitor cocktail
1M buffer
- 10 mM Tris-HCl (pH 7.3)
- 0.5 M sucrose
- 40 µM CaCl₂
- 0.23 mM PMSF
- 1 mM DTT
DDM/CHS solution
- 5g DDM + 0.5g CHS, dissolved in 50 ml milliQ water
Wash buffer 1
- 20 mM Tris-HCl (pH 7.5)
- 300 mM NaCl
- 0.03% DDM
- 0.003% CHS
- 5 mM TCEP
Wash buffer 2
- 20 mM Tris-HCl (pH 7.5)
- 150 mM NaCl
- 0.03% DDM
- 0.003% CHS
- 5 mM TCEP
Elution buffer
- 100 mM Tris-HCl (pH 8.0)
- 150 mM NaCl
- 1 mM EDTA
- 50 mM biotin
- 0.03% DDM
- 0.003% CHS
- 5 mM TCEP
Materials
- 500 cm2 dishes (Cat# 734-1727; VWR)
- WHEATON‱ Dounce Tissue Grinder, 40 mL (Cat# 357546; DWK Life Sciences)
- n-dodecyl-β-D-maltoside (DDM) (Cat# 1758-1350; Inalco)
- Cholesteryl hemisuccinate (CHS) (Cat# C6013; Sigma-Aldrich)
- Tris: Trizma base (Cat# T1503; Sigma-Aldrich)
- HCl (Cat# 1.00317.2501; Merck)
- EDTA (Ethylenediaminetetra-acetic acid) (Cat# 280214S; BDH Laboratory Supplies)
- NaCl (Cat# 0962.2; Carl Roth)
- Strep-Tactin‱XT 4Flow‱ high-capacity resin (Cat# 2-5030-002; IBA LifeSciences)
- Biotin (Cat# 2-1016-002; IBA LifeSciences)
- Bond-Breaker‱ TCEP Solution (Cat# 77720; Thermo Fisher)
- MgCl₂ (Cat# M2670; Sigma-Aldrich)
- DTT: Dithiothreitol (Cat# A2948.0025; VWR)
- phenylmethylsulfonyl fluoride (PMSF) (Cat# 93482; Sigma-Aldrich)
- SigmaFast‱ Protease Inhibitor Cocktail (Cat# S8830; Sigma-Aldrich)
- sucrose (Cat# S7903; Sigma-Aldrich)
- CaCl2 (Cat# C3881; Sigma-Aldrich)
- Nanodrop spectrophotometer (Thermo Fisher)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Cell collection
1d 5h 53m
Seed HEK293T cells (with stable ATP13A4 expression, generated via lentiviral transduction) into 500 cm2 dishes at a density appropriate to achieve 70-80% confluency by the following day. Allow the cells to attach and grow for approximately 24:00:00 .
Note
Protocol to generate stable cell lines via lentiviral transduction:
1d
The following day, gently detach and harvest the cells by scraping them into PBS.
Spin cells down (400 x g, 00:05:00 ).
5m
Resuspend cell pellet in PBS.
Spin cells down (400 x g, 00:05:00 ).
5m
Cell lysis
1d 5h 53m
Resuspend cell pellet in 25 mL LIS buffer and keep 00:15:00 on ice.
15m
Transfer to Dounce homogenizer and apply 60 up-and-down strokes on ice.
Add 25 mL 1M buffer, apply 30 up-and-down strokes on ice.
Centrifuge for 00:20:00 at 2000 rpm, 4°C . Keep supernatant.
20m
Resuspend resulting cell pellet in 25 mL LIS buffer and keep 00:15:00 on ice.
15m
Transfer to Dounce homogenizer and apply 60 up-and-down strokes on ice.
Add 25 mL 1M buffer, apply 30 up-and-down strokes on ice.
Centrifuge for 00:20:00 at 2000 rpm, 4°C . Keep supernatant.
20m
Combine the supernatants of and
Determine protein concentration of supernatant via nanodrop.
Membrane solubilization
1d 5h 53m
Add DDM (supplemented with CHS at a DDM:CHS ratio of 10:1) at three times the amount of protein concentration in the supernatant.
Note
For example, if the protein concentration of the supernatant is 10 mg/mL, add DDM to achieve a final concentration of 30 mg/mL.
Solubilize for 03:00:00 at 4 °C in a head-over-head rotator.
3h
Spin at 11000 rpm, 4°C for 01:00:00 .
1h
Affinity purification
1d 5h 53m
Equilibrate Strep-Tactin®XT resin by washing it with SSR buffer (10 column volumes; 300 x g, 4°C , 00:03:00 ).
3m
Add 100 µL prewashed Strep-Tactin®XT beads to the supernatant ( ), bind overnight in head-over-head rotator at 4 °C
Pour the mixture into a large column and allow it to drip through by gravity flow.
Wash resin twice with 10 column volumes wash buffer 1.
Wash resin twice with 10 column volumes wash buffer 2.
Elute ATP13A4 with SSR buffer supplemented with 50 mM biotin (E0-6).
Elution 0: add 150 µl elution buffer and collect the eluate (E0).
Elution 1: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E1).
5m
Elution 2: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E2).
5m
Elution 3: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E3).
5m
Elution 4: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E4).
5m
Elution 5: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E5).
5m
Elution 6: add 150 µl elution buffer, incubate for 00:05:00 in head-over-head rotator at 4 °C and collect the eluate (E6).
5m
Quality control
1d 5h 53m
Analyze purification quality using SDS-PAGE followed by Coomassie staining or immunoblotting.
Estimate protein concentration by running an SDS-PAGE gel with BSA standards and staining with Coomassie dye.