Jun 15, 2026

SDS-PAGE and western blot analysis

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvje64wgk5/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvje64wgk5/v1
Protocol Citation: Elisabeth Holzer 2026. SDS-PAGE and western blot analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvje64wgk5/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
-
Created: July 31, 2025
Last Modified: June 15, 2026
Protocol  Integer ID: 223987
Keywords: ASAPCRN, western blot analysis technique, western blot analysis this protocol, western blot analysis, sd
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol describes the SDS-PAGE and western blot analysis technique.
Materials
  • NuPAGE™ 4 to 12% Bis-Tris 1.0 mm Mini Protein Gel 10-wellInvitrogen - Thermo FisherCatalog #NP0321BOX or
  • NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-wellThermo FisherCatalog #NP0323BOX
  • PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDaThermo FisherCatalog #26620 or
  • PageRuler™ Prestained Protein Ladder, 10 to 180 kDaThermo FisherCatalog #26617
  • Nitrocellulose membranes (GE Healthcare, Cat# RPN132D)
  • Mini Trans-Blot Cell (Bio-Rad, Cat# 20-45-111)
  • TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
  • SuperSignal™ West Femto Maximum Sensitivity SubstrateThermo FisherCatalog #34096

Primary antibodies:

  • Atg9A (D4O9D) Rabbit mAbCell Signaling TechnologyCatalog #13509
  • GABARAP (E1J4E) Rabbit mAbCell Signaling TechnologyCatalog #13733
  • LC3B (D11) XP® Rabbit mAbCell Signaling TechnologyCatalog #3868
  • Anti-p62/SQSTM1BD BiosciencesCatalog #610833

Secondary antibodies:

  • Anti-Mouse IgG (H L) Goat Polyclonal Antibody (HRP (Horseradish Peroxidase))Jackson ImmunoResearch Laboratories, Inc.Catalog #115-035-003
  • Peroxidase AffiniPure® Goat Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-035-003

Gel Electrophoresis and Protein Transfer
1h 30m
Load samples onto 4–12% SDS-PAGE gels (Invitrogen, Cat# NP0321BOX or NP0323BOX).
Include PageRuler Prestained protein marker (Thermo Fisher, Cat# 26620 or 26617) for size reference.
Run the gel according to standard electrophoresis protocols.
Transfer proteins onto nitrocellulose membranes (GE Healthcare, Cat# RPN132D) for 01:30:00 using the Mini Trans-Blot Cell (Bio-Rad, Cat# 20-45-111).

1h 30m
Blocking and Antibody Incubation
3h 5m
Block membranes for 01:00:00 at Room temperature with 5% milk powder in TBS-T.
1h
TBS-T: 0.1% Tween 20 (Sigma-Aldrich, Cat# P7949-500ML).
Incubate membranes Overnight at 4 °C with primary antibodies diluted in blocking buffer.
1h
Wash membranes three times for 00:05:00 each with TBS-T.
5m
Incubate with HRP-conjugated secondary antibodies diluted 1:10,000 in blocking buffer for 01:00:00 at Room temperature .
1h
Wash membranes three times with TBS-T.
Detection and Imaging
Incubate membranes with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Cat# 34096).
Image blots using ChemiDoc MP Imaging System (Bio-Rad).
Analyze the images using ImageJ (RRID:SCR_003070; https://imagej.net/).