Mar 19, 2025

Public workspaceGel Electrophoresis and Immunoblotting

DOI
dx.doi.org/10.17504/protocols.io.n92ldro4ng5b/v1
  • Patricia Yuste-Checa1,
  • Andreas Bracher1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Patricia Yuste-Checa
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DOI: dx.doi.org/10.17504/protocols.io.n92ldro4ng5b/v1
Protocol Citation: Patricia Yuste-Checa, Andreas Bracher, F Ulrich Hartl 2025. Gel Electrophoresis and Immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldro4ng5b/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: March 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124383
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to separate proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Native PAGE, and subsequent detection by immunoblotting.
Materials
Buffers, reagents and consumables:

  • NuPAGE 4–12% Bis-Tris SDS gels (Thermo Fisher Scientific)
  • NuPAGE MES SDS running buffer (20X) (Thermo Fisher Scientific)
  • NuPAGE™ LDS Sample Buffer (4X) (Thermo Fisher Scientific)
  • Pre-stained molecular weight SDS-PAGE marker (PageRuler Prestained Protein Ladder, 10 to 180 kDa, ThermoFisher)
  • ReagentNativePAGE 3%–12% Bis-Tris SDS gel Thermo Fisher ScientificCatalog #BN1001BOX
  • ReagentNativePAGE™ Running Buffer (20X)Thermo FisherCatalog #BN2001
  • ReagentNativePAGE™ Sample Buffer (4X)Thermo FisherCatalog #BN2003
  • ReagentNativeMark™ Unstained Protein StandardThermo FisherCatalog #LC0725
  • Select Western blot transfer stacks nitrocellulose (Invitrogen)
  • Tris-buffered saline (TBS, Concentration10 millimolar (mM) Tris-HCl Ph7.5 , Concentration150 millimolar (mM) NaCl) containing 0.05% Tween 20 (0.05% TBS-T)
  • ReagentPower Blotter 1-Step™ Transfer Buffer (5X)Thermo FisherCatalog #PB7100
  • PBS-T (1x PBS with 0.1% Tween 20)
  • 3% bovine serum albumin (BSA) (Merck) in TBS-T. Store at Temperature4 °C .
  • ReagentPower Blotter Select Transfer Stacks, nitrocellulose, miniThermo FisherCatalog #PB3210
  • ReagentPower Blotter 1-Step™ Transfer Buffer (5X)Thermo FisherCatalog #PB7100

Instruments

  • Microcentrifuge for 1.5 ml reaction tubes (Eppendorf)
  • Semi-dry blotting apparatus (Power Blotter XL, Invitrogen)
  • Luminescence gel documentation system (ImageQuant LAS 4000 mini, General Electric)




Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Prepare the samples by mixing with NuPAGE™ LDS Sample Buffer (4X) supplemented with Concentration100 millimolar (mM) DTT final concentration.

Pipetting
Mix
Boil the samples for Duration00:05:00 .

5m
Brief centrifugation to make sure nothing remains in the lid due to condensation.
Centrifigation
Load the samples into the pockets of a NuPAGE 4–12% Bis-Tris SDS gel, or similar and run using NuPAGE MES SDS running buffer at 140 V.
Pipetting
Native-PAGE
Native-PAGE
Prepare the samples by mixing with NativePAGE Sample Buffer (4X).
Pipetting
Mix
Load the samples into a NativePAGE 3–12% Bis-Tris SDS gel or similar and run using NativePAGE running buffer at 140 V.
Pipetting
Mix
Protein Transfer to a Membrane
Protein Transfer to a Membrane
Remove carefully the gel from the case. Native-PAGE gel are especially sticky and susceptible to rip.
Wash the gel briefly with de-ionized water.
Wash
Perform a semi-dry transfer by placing the gel on top of the nitrocellulose membrane provided in Power Blotter Select Transfer Stacks, nitrocellulose, mini. Wet the stack with some 1x transfer buffer (Power Blotter 1-Step Transfer Buffer 5X) and make sure to remove all bubbles using a roller.
Place on top the filter paper provided with the stack and remove bubbles using a roller.
Place the stack including your gel into an Invitrogen Power Blotter semi-dry transfer system.
Set the corresponding program depending on the number of gels to be transferred and the size of the proteins of interest.
Once the transfer is done, wash the membrane with Tris Buffer Saline, 0.05% Tween (TBS-T).
Wash
Block the membrane during at least Duration01:00:00 with blocking buffer: TBS-T 5% milk or TBS-T 3% bovine serum albumin.

1h
After blocking, incubate the membrane with the primary antibody in blocking buffer. Depending on the antibody, incubate from Duration01:00:00 -Duration02:00:00 at TemperatureRoom temperature to DurationOvernight at Temperature4 °C .

2h
Incubation
Overnight
Temperature
Wash the membrane for Duration00:10:00 with TBS-T. Repeat washing three times.

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (1/3).

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (2/3).

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (3/3).
10m
Wash
Incubate with corresponding horseradish peroxidase (HRP) conjugated secondary antibody in blocking buffer during Duration02:00:00 at TemperatureRoom temperature .

2h
Incubation
Temperature
Wash the membrane for Duration00:10:00 with TBS-T. Repeat washing three times.

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (1/3).

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (2/3).

10m
Wash
Wash the membrane for Duration00:10:00 with TBS-T (3/3).
10m
Wash
Place the membrane in the developer tray of an imaging system like the Amersham ImageQuant 800.
Develop the membrane by addition of enhanced chemiluminescence (ECL) Western blot substrate like Immobilon Forte Western HRP substrate (Merck).
Remove the excess of substrate and record image.