Jan 28, 2026
Western Blot of human iPSC-derived Dopamine Neurons (DaNs)
Forked from Immunoblots
- Kaitlyn Cramb1,2,3,4,
- Iona Thomas-Wright1,2,3,4,
- Richard Wade-Martins1,2,3,4
- 1Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Sherrington Rd, Oxford, OX1 3PT, UK;
- 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, UK;
- 3Oxford Parkinson’s Disease Centre, University of Oxford, Oxford OX1 3PT, UK;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Protocol Citation: Kaitlyn Cramb, Iona Thomas-Wright, Richard Wade-Martins 2026. Western Blot of human iPSC-derived Dopamine Neurons (DaNs). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8obxv5b/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: October 16, 2024
Last Modified: January 30, 2026
Protocol Integer ID: 110035
Keywords: western blot of human ipsc, derived dopamine neuron, dopamine neuron, standard procedure for protein separation, protein separation, synuclein, identification of protein, human ipsc, similar to other western blot procedure, protein, other western blot procedure, western blot, ipsc, dan
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020370, ASAP-025192
Abstract
This protocol describes a standard procedure for protein separation and identification of protein of interest. The steps are similar to other Western Blot procedures found elsewhere with small modifications to prevent aggregation or post-transfer loss of alpha-synuclein.
Materials
Reagents:
- Gel, 4-5%, 18 wells, Stain-free Midi-PROTEAN Precast Gels (Bio-Rad Laboratories Ltd, CAT# 5678084)
- Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs (Bio-Rad Laboratories Ltd, CAT# IPVH000101704156)
- Tris base (ThermoFisher Scientific, CAT# BP152-5)
- Glycine (ThermoFisher Scientific, CAT# BP381-5)
- Spectra™ Multicolor Broad Range Protein Ladder (ThermoFisher Scientific, CAT# 26634)
- TBS
- Triton X100 (Sigma-Aldrich, CAT# T8787-250ML)
- Protease inhibitor (complete Mini, EDTA-free, Roche)
Primary Antibodies:
- Rabbit anti-α-synuclein [MJFR1] (Abcam, CAT# ab138501, dilution 1:10000)
- Rabbit anti-DDC (Sigma-Aldrich, CAT# AB1569, dilution 1:1000)
- Rabbit anti-MAO-B (Abcam, CAT# ab137778, dilution 1:1000)
- Rabbit anti-TH (Millipore, CAT# AB152, dilution 1:1000)
- Rabbit anti-Phosphorserine TH (Sigma-Aldrich, CAT# AB5935, dilution 1:1000)
- Rabbit anti-vATPase (Abcam, CAT# Ab179858, dilution 1:1000)
- Rabbit anti-vGLUT2 (Synaptic Systems, CAT# 135403, dilution 1:1000)
- Rabbit anti-VMAT2 (Abcam, Ab70808, dilution 1:1000)
Secondary Antibody:
- Donkey anti-Rabbit IgG Alexa Fluor 647 (Molecular Probes, RRID:AB_2536183)
SOLUTIONS
Preparing RIPA buffer:
- 10 mM Tris-HCl
- 1 mM EDTA
- 1 mM EGTA
- 140 mM NaCl
- 1% Triton X-100
- 0.1% SDS
- 0.1% Sodium deoxycholate pH 7.4
Preparing TBS-T:
- 20 mM Tris pH 7.4
- 150 mM NaCl
- 0.1% Tween-20
Troubleshooting
Sample Preparation
4h 50m
Lyse cells in RIPA buffer (see Materials) with protease inhibitor for 30 minutes and spun 10 min 10,000x g to remove cell debris.
45m
Mix protein-containing supernatants with SDS sample loading buffer and 50 mM DTT.
Note
We use 10 or 20 μg proteins per lane depending on the target of interest.
10m
Gel Electrophoresis and Membrane Transfer
Heat samples at 75°C for 10 min to prevent aggregation.
Separate samples on SDS-PAGE gels for 45 mins, 200 V.
Image separated proteins for total protein using the stain-free diazo dye.
Transfer separated proteins to PVDF membranes using the Trans-Blot Turbo Transfer System.
Fixation, Blocking and Antibody Incubation
30m
Fix membranes for 30 min in 4% formaldehyde solution to prevent loss of small proteins.
30m
Block membranes with 5% skimmed milk powder in TBS-T (see Materials) at room temperature for 1 h with constant agitation.
Incubate membranes with the desired Primary Antibody (see Materials) at 4°C overnight.
Wash membranes with TBS-T for 3 times (10 mins each) with agitation.
Incubate membranes with secondary antibody for 1 h at room temperature.
Detection
Use Amersham ECL substrate and image membrane using BioRad ImageLab.