Feb 03, 2025
Western blot
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk94x5v5r/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112825
Funders Acknowledgements:
ASAP
Abstract
Western blot
For cell experiments
For cell experiments
Extract proteins using 0.5% NP40–PBS protein extraction buffer containing protease and phosphatase inhibitors (Roche, Switzerland) on ice.
Centrifuge the suspension at 14,000 rpm for 15 minutes at 4°C
For mouse tissue experiments
For mouse tissue experiments
Extract and clean tissue with PBS.
Dissect the cortex quickly to prevent protein degradation.
Add ice-cold homogenization buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and protease and phosphatase inhibitors) at approximately 1 mL per gram of tissue.
Homogenize the tissue manually with a pestle for 10–20 passes on ice.
Centrifuge the homogenate at 14,000 rpm for 20 minutes at 4°C.
After centrifugation for both cell and mouse tissue experiments
After centrifugation for both cell and mouse tissue experiments
Measure protein concentration in the supernatant using the Pierce‱ BCA Protein Assay Kit (Cat. number 23225, Thermo Fisher Scientific).
Load 20–50 µg of protein lysate onto a polyacrylamide gel (SurePAGE‱, Bis-Tris, 10x8, 4%–12%, GenScript).
Separate proteins via electrophoresis and transfer them onto a 0.45 µm polyvinylidene fluoride (PVDF) membrane (Millipore).
Block the membrane with either 5% milk powder or 5% bovine serum albumin (BSA) in TBS containing 0.1% Tween 20 (TBST).
Incubate the membrane overnight at 4°C with primary antibodies diluted in the chosen blocking solution.
Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma‒Aldrich) for 1 hour at room temperature.
Visualize the proteins using ECL Western Blotting Detection Reagent (Pierce, Thermo Fisher Scientific) in one of the following imaging systems:
- Odyssey DLx Imager (LI-COR),
- Stella imaging system (Raytest), or
- ChemiDoc MP imaging system (Bio-Rad).
Perform densitometric analysis of protein expression using ImageJ software.