Jan 16, 2026
Western Blotting Protocol
- Mannan Nouri1,2
- 1Harvard Medical School;
- 2Beth Israel Deaconess Medical Center
Protocol Citation: Mannan Nouri 2026. Western Blotting Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1mzol5b/v1
Manuscript citation:
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: January 13, 2026
Last Modified: January 16, 2026
Protocol Integer ID: 238583
Keywords: Western blotting, immunoblotting, protein extraction, whole-cell lysate preparation, lysate clarification, protein quantification, BCA assay, protein normalization, sample denaturation, SDS-PAGE, gel electrophoresis, protein transfer to membrane, rapid transfer system, membrane blocking, membrane washing, primary antibody incubation, secondary antibody incubation, chemiluminescent detection, fluorescent detection, film exposure, digital imaging, phosphoprotein detection, membrane sectioning, re-probing, membrane storage, membrane transfer for western blot detection, cell protein lysates by ripa lysi, cell protein lysate, western blot detection, protein by bca, western blotting protocol, phosphorylation shifts after short serum, quantifying protein, western blotting protocol this protocol, membrane transfer, phosphorylation shift, ripa lysi, secondary antibody, membrane, serum
Abstract
This protocol describes culturing adherent cells, harvesting whole-cell protein lysates by RIPA lysis after drug or transfection treatments, quantifying protein by BCA, and performing SDS-PAGE and membrane transfer for Western blot detection. Membranes are blocked and probed with primary and secondary antibodies, followed by chemiluminescent (or fluorescent) visualization and optional re-probing.
Expected results are consistent, comparable protein inputs across conditions (normalized to concentration) and clear, well-resolved target bands at the expected molecular weight with minimal background. Treatment-dependent changes (e.g., phosphorylation shifts after short serum-starve/drug exposure or expression changes after longer treatments) should be detectable as reproducible differences in band intensity or mobility between conditions.
Materials
- 0.25% Trypsin (Fisher, #MT25053CI)
- 10% Fetal Bovine Serum/FBS (Fisher, #SH3007203)
- 10% Charcoal Stripped Serum/CSS (Fisher, #SH3006803)
- Trypan Blue Solution (Fisher, #MT-25-900-CI)
- 1X PBS (Fisher, #4054-1012)
- RIPA (Fisher, #89901)
- Phosphatase inhibitor (Fisher, #78426)
- Protease inhibitor (Fisher, #78439)
- 1.5mL Eppendorf tube
- p200 pipette
- BCA analysis kit (Fisher, #PI23225)
- 4X Laemmli buffer (BioRad, #1610747)
- β-Mercaptoethanol
- Gel apparatus
- Running buffer (10x Tris/Glycine/SDS, Bio-Rad, #1610772)
- Gel loading tip (USA Scientific, #1022-0600)
- Pre-cast 4-20% 15-Well gradient gels (BioRad, #4561093EDU)
- Precision Plus Dual Color Ladder (BioRad, #1610394)
- Trans-Blot Turbo Transfer system (BioRad)
- Trans-Blot Turbo RTA Mini 0-2 um Nitrocellulose Transfer Kit (#1704270)
- 5% milk (Fisher, #NC9121673)
- 1X Tris Buffered Saline (BioRad, #1706435)
- Tween20 (Sigma-Aldrich, P1379-500ML)
- TBS-T
- 5% BSA (Fisher, AM2616)
- Kimwipe (Fisher, 06666A)
- Chemiluminescence reagents (Fisher, #509049325)
- Film (Research Products International Corp, #248300)
- Electronic imagers (BioRad/Licor)
Troubleshooting
Safety warnings
- Under/over seeded cells will result in aberrant results.
- Serum starve and treat for 30 minutes for phosphorylation targets.
- If not moving immediately to analysis, lysates can be stored at -20°C.
- Debris pellet can be removed with p20 pipette tip or the supernatant can be transferred to another tube to avoid contamination of protein lysate with residual debris.
- Use loading buffer and β-Mercaptoethanol to top up any wells with discordant volumes.
- In lieu of overnight antibody incubation, membranes may be probed for one hour at room temperature with a loss in band quality/definition and an increase in background.
- Do not blot the protein side, or allow the membrane to fully dry.
- Do not stack membranes in container, or allow them to contact the protein side of any other membrane(s).
Cell Culture and Harvest
Dissociate adherent cells with 0.25% Trypsin (Fisher, #MT25053CI) for 5-10 minutes.
Deactivated Trypsin in 10% Fetal Bovine Serum/FBS (Fisher, #SH3007203) or 10% Charcoal Stripped Serum/CSS (Fisher, #SH3006803) media, and pellet cells at 1500 RPM.
Re-suspend cells in culture medium, and count employing Trypan Blue Solution (Fisher, #MT-25-900-CI) and manual or electronic Hemacytometer.
Seed cells into individual wells of a 6-Well plate for all conditions equally and allow appropriate time for attachment (eg: 24-48 hours).
- Note: Under/over seeded cells will result in aberrant results, and seeding densities should be determined ahead of time to prevent harvest of over-confluent, clumpy or detached cells.
Treat cells with drug, or transfection reagents according to manufacturer’s instructions.
Note: Serum starve and treat for 30 minutes for phosphorylation targets, and treat for 24-96 hours with our without starvation for other targets.
Wash cells gently with 1X PBS (Fisher, #4054-1012) and conduct protein harvest in 120µL of RIPA (Fisher, #89901) supplemented with phosphatase inhibitor (Fisher, #78426) and protease inhibitor (Fisher, #78439), by scraping cells and transferring to a 1.5mL Eppendorf tube with a p200 pipette.
Incubate samples for 30 minutes at 4°C to allow lysis to proceed.
- Note: If not moving immediately to analysis, lysates can be stored at -20°C.
After 30 minute incubation, or thaw from freezing storage, spin down samples at max RPM at 4°C for 10 minutes to remove cell debris before protein quantification.
- Note: Debris pellet can be removed with p20 pipette tip or the supernatant can be transferred to another tube to avoid contamination of protein lysate with residual debris.
Western Blot
Conduct Bicinchoninic Acid (BCA) analysis, to determine protein lysate concentrations according to manufacturer’s instructions (Fisher, #PI23225).
Dilute all samples in RIPA and 4X Laemmli buffer (BioRad, #1610747) to a total volume of 100µL at 1µg/µL.
- Note: Be sure to add β-Mercaptoethanol to Laemmli right before addition to working samples.
Heat samples for 5 minutes at 95°C to denature most proteins. Alternatively, for heat-sensitive proteins, membrane proteins, or when you need to preserve protein interactions, heat samples for 10 minutes at 65°C to denature.
Prepare gel apparatus, and employ appropriate running buffer (10x Tris/Glycine/SDS, Bio-Rad, #1610772, recommended).
Use a gel loading tip (USA Scientific, #1022-0600) to wash wells of gel with running buffer to remove storage buffer.
Load samples onto gel (BioRad pre-cast 4-20% 15-Well gradient gels [#4561093EDU] preferred), again using a gel loading tip.
Ensure all wells have the same volume to prevent band malformations.
- Note: Use loading buffer and β-Mercaptoethanol to top up any wells with discordant volumes. eg: 6µL of Loading Buffer and 4µL of Precision Plus Dual Color Ladder (BioRad, #1610394), or 10µL of Loading Buffer in an empty well, to make all wells 10µL.
Run gel(s) at 5 milliamps constant per gel for 30 minutes to allow samples to enter gel, then run at 10-50 milliamps constant until dye front leaves gel, or until desired separation occurs.
Transfer gel to membrane using BioRad Trans-Blot Turbo Transfer system along with the Trans-Blot Turbo RTA Mini 0-2 um Nitrocellulose Transfer Kit (#1704270) according to manufacturer’s instructions (typically 1.3amps and 25 volts for 7 minutes for mixed molecular weight targets).
Block membranes in 5% milk (Fisher, #NC9121673) in 1X Tris Buffered Saline (BioRad, #1706435) plus 0.1% Tween20 (Sigma-Aldrich, P1379-500ML) (TBS-T) for 60 minutes on a shaker immediately after transfer completes.
- Note: For detection of phosphorylated-proteins blocking in 5% BSA TBS-T is recommended.
Probing Western Membranes
Wash whole or sectioned membranes with TBS-T to remove Milk.
Incubate membranes with appropriate primary antibody dilution in 5% BSA (Fisher, AM2616) in TBS-T overnight on a shaker at 4°C.
- Note: In lieu of overnight antibody incubation, membranes may be probed for one hour at room temperature with a loss in band quality/definition and an increase in background.
Wash membranes with TBS-T three times for a total of 15 minutes.
Incubate membranes with appropriate secondary antibody dilution in 5% Milk/BSA TBS-T for one hour on a shaker at 4°C.
- Note: If using fluorescent secondary antibodies, cover membranes to protect from light during incubation and washes.
Wash membranes with TBS-T three times for a total of 15 minutes.
- Note: If using fluorescent secondary antibodies go directly to imaging in a Li-Cor Odyssey or other imager.
Place membranes protein side up on a Kimwipe (Fisher, 06666A) to blot excess buffer, and allow to dry for 1-3 minutes.
- Note: Do not blot the protein side, or allow the membrane to fully dry.
Prepare chemiluminescence reagents (Fisher, #509049325) in a container large enough to fit membrane(s).
Incubate the membranes in chemiluminescence reagents for 1 minute with gentle agitation.
- Note: Do not stack membranes in container, or allow them to contact the protein side of any other membrane(s).
Expose membranes using film (Research Products International Corp, #248300) or electronic imagers (BioRad/Licor).
Wash membranes with TBS-T two times to remove chemiluminescence reagents.
Membranes can be stored at 4°C in TBS (not TBS-T), or re-probed for other targets immediately.