Apr 15, 2026

Immunoblotting

DOI
https://dx.doi.org/10.17504/protocols.io.14egn96rql5d/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.14egn96rql5d/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn96rql5d/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 11, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 115027
Keywords: ASAPCRN, Immunoblotting, Western blot, ATP13A4, protein separation by gel electrophoresi, performing western blot analysis, western blot analysis, gel electrophoresi, antibody incubation, protein separation, including protein separation, antibody, electrophoresi, western blot, protein
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol outlines a step-by-step method for performing western blot analysis, including protein separation by gel electrophoresis, transfer to membranes, antibody incubation, and detection.
Materials
Buffers
TBS-T
  • 50 mM Tris-HCl (pH 7.5)
  • 150 mM NaCl
  • 0.1% Tween-20

Materials
  • β-mercaptoethanol (Cat# M3148, Sigma)
  • Anti-mouse IgG, HRP-linked Antibody (Cat# 7076, RRID:AB_330924, Cell Signaling Technology)
  • Anti-rabbit IgG, HRP-linked Antibody (Cat# 7074, RRID:AB_2099233, Cell Signaling Technology)
  • BSA (Cat# A4161, Sigma)
  • HCl (Cat# 1.00317.2501, Merck)
  • Mouse anti-GAPDH (Cat# G8795, RRID:AB_1078991, Sigma)
  • Mouse anti-Flag-tag (Cat# MA1-91878, RRID:AB_1957945, Thermo Fisher Scientific) 
  • Mouse anti-streptavidin (Cat# ab76949, RRID:AB_1524455, Abcam)
  • NaCl (Cat# 0962.2, Carl Roth)
  • NuPAGETM LDS Sample Buffer (4X) (Cat# NP0007, Invitrogen)
  • NuPAGETM 4–12% Bis-Tris precast gels (Cat# NP0321, Cat# NP0322, Cat# NP0323; Invitrogen)
  • NuPAGETM MOPS SDS Running Buffer (20X) (Cat# NP0001; Invitrogen)
  • NuPAGETM Transfer Buffer (20X) (Cat# NP00061; Invitrogen)
  • PierceTM ECL Western Blotting Substrate (Cat# 32106, Thermo Fisher)
  • PVDF membrane (Cat# IPFL00010, Millipore)
  • Rabbit anti-ATP13A4 (PMID: 37371498)
  • SeeBlueTM Plus2 Pre-stained Protein Standard (Cat# LC5925, Invitrogen)
  • Tris (Cat# T1503, Sigma)
  • Tween-20 (Cat# A4974.0500, PanReac AppliChem)

Equipment
  • Centrifuge
  • Bio-Rad ChemiDoc MP imaging system
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Protein separation by gel electrophoresis
1h 5m
Prepare protein samples with LDS sample loading buffer (supplemented with β-mercaptoethanol).
Boil samples for 00:05:00 at 95 °C .
5m
Load equal amounts of protein (total cell lysate: 20-40 µg protein, purified protein: 1 ug) into the wells of a precast NuPAGE 4-12% BisTris gel, alongside molecular weight markers for reference.
Run the gel for 01:00:00 at 170 V, Room temperature . Use MOPS running buffer.     

1h
Transferring the protein from the gel to the membrane
1h 0m 5s
Activate polyvinylidene fluoride (PVDF) membrane in methanol (00:00:05 ).

5s
Assemble transfer sandwich and make sure no air bubbles are trapped in the sandwich.
Place the cassette in the transfer tank with an ice block to maintain low temperature. Fill with transfer buffer containing 20% methanol.
Transfer for 01:00:00 at 100 V (or alternatively, Overnight at 30 V) in a cold room.

1h
Antibody incubation
3h 55m
Block 01:00:00 in TBS-T supplemented with 5% non-fat dry milk, at Room temperature

1h
Incubate the blot with primary antibodies (01:00:00 ; 1:1,000 dilution in TBS-T, 1% BSA), Overnight at 4°C.
2h
Wash the blot 3 times for 00:05:00 in TBS-T, at Room temperature .

5m
Incubate the blot with HRP-conjugated secondary antibodies (00:45:00 ; 1:1,000 dilution in TBS-T, 1% milk), at Room temperature .

45m
Wash the blot 3 times for 00:05:00 in TBS-T, at Room temperature .

5m
Detection
Apply enhanced chemiluminescence (ECL) substrate to the blot and detect protein signals using the Bio-Rad ChemiDoc MP imaging system.
Quantification
Analyze protein band intensity using ImageJ software (http://fiji.sc; RRID:SCR_002285).
Normalize protein levels to loading controls (e.g. GAPDH).
Protocol references
van Veen S, Kourti A, Ausloos E, Van Asselberghs J, Van den Haute C, Baekelandt V, et al. ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7. Biomolecules. 2023;13(6).