Jan 10, 2026
Protein analyses and immunoblotting
- Ali Ghoochani1,2,3,4,
- Monther Abu-Remaileh1,2,3,4
- 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
- 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
- 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- asap
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Protein analyses and immunoblotting. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbw15ngpk/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: July 08, 2025
Last Modified: January 10, 2026
Protocol Integer ID: 222033
Keywords: complete workflow for western blotting, western blotting, quantification of target protein, protein quantification, antibody incubation, including cell lysi, antibody, cell lysi, protein transfer, target protein, protein
Abstract
This protocol describes the complete workflow for Western blotting, including cell lysis, protein quantification, SDS-PAGE, protein transfer, antibody incubation, and detection . The method allows for the detection and quantification of target proteins.
Troubleshooting
Cell Lysis
Prepare 1X lysis buffer:
- 50mM HEPES pH: 7.4
- 40mM NaCl
- 2mM EDTA
- 1% Triton X
- 1.5 mM NaVo4
- 300mM NaF
- 10mM Napyrophosphate
- 10mM NaBetaGlycerophosphate
Store lysis buffer at 4C.
Add:
• Add 1 tablet cOmplete‱ Mini Protease Inhibitor Cocktail for 10 ml lysis buffer
• Add 1 tablet PhosSTOP‱ Phosphatase Inhibitor for 10 ml lysis buffer
Partial cell use:
• Trypsinize and harvest cells into tubes on ice
• Centrifuge at 1000 x g, 4°C , 2 min
• Resuspend in 100–200 µL lysis buffer (6-well) or 300–500 µL (10 cm dish)
Use all cells:
• Place plate on ice, aspirate media, and wash with cold PBS
• Add 100–200 µL lysis buffer per well, scrape, and collect in tubes on ice
• Incubate on shaker at 4°C for 15–30 min
• Centrifuge at max speed, 4°C, 10 min
• Transfer supernatant and normalize protein concentration
SDS-PAGE
•Select gel percentage based on protein size (e.g., 8% for >100 kDa, 12% for >20 kDa)
• Prepare 1× SDS running buffer and rinse tank
• Load 5 µL ladder and up to 30 µL of each sample (5 to 30µg proteins)
• Run at 120 V for ~150 min.
Gel Transfer
• Prepare transfer buffer and soak PVDF membrane (0.45 µm) in ethanol, then transfer buffer.
• Assemble membrane-gel sandwich in cassette
• Place in tank filled with transfer buffer
• Transfer at 40 V for 2 hours at room temperature
Blocking and Antibody Incubation
• Block membranes in 5% bovine serum albumin (BSA) in TBS-T (Tris-buffered saline with Tween-20) for 30–60 min
• Incubate with primary antibody in 5% BSA overnight at 4°C.
• Wash membranes 3× with TBST
• Incubate with secondary antibody (1:3000 in 5% milk) for 1–1.5 h
• Wash 3× for a total of 30 min.
Detection
• Mix equal parts of ECL reagents (Pierce)
• Incubate membrane in reagent for 1–10 min
• Visualize membrane with any Chemidoc imaging system.
Buffers:
To make 10 L of SDS running buffer:
- 300g Tris base
- 1440g Glycine
- 100g SDS
Recipe for 10x TBS-T
- 20L of 10x TBS-T
- 1.37 M NaCl, 200mM Tris, 1% Tween, pH 7.6
- 1,601.26 g NaCl
- 501.2 g Tris HCl
- 200 gTris Base
- 200 mL Tween
Transfer buffer:
- 15 NaOH pellets (americanbio #AB1916-01000)
- 8.8g CAPS (Sigma #C6070-1KG)
- 400 mL ethanol
- 3600 mL deionized water
1X lysis buffer:
- 50mM HEPES pH: 7.4
- 40mM NaCl
- 2mM EDTA
- 1% Triton X
- 1.5 mM NaVo4
- 300mM NaF
- 10mM Napyrophosphate
- 10mM NaBetaGlycerophosphate
Store lysis buffer at 4C.