Jan 14, 2026
Western blot
- Yongjia Duan1
- 1University of California Berkeley
Protocol Citation: Yongjia Duan 2026. Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bprzl3e/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: January 14, 2026
Last Modified: January 14, 2026
Protocol Integer ID: 238670
Keywords: ASAPCRN, western blot
Abstract
Western blot
Troubleshooting
Cell lysis
Cells were lysised with 2% SDS extraction buffer ((50mM Tris pH 6.8, 2% SDS, 1%
mercaptoethanol, 12.5% glycerol, 0.04% bromophenol blue, and the protease
inhibitor cocktail (Roche, 04693132001)).
Then, the mixture were boiled at 95 °C for 10min. Then, the sample were spin down and were ready for the next step.
SDS-Page
Then sample were separated with 4-12% Bis-Tris SDS-PAGE or 6–14% Bis Tris gels. Then, proteins were transferred onto nitrocellulose (NC) membranes using a semi-dry transfer system at 25mA for 60 minutes.
Membranes were blocked with 5% non-fat milk in TBST (TBS containing 0.1% Tween-20) for 1 hour at room temperature with gentle shaking. Then, were incubated with primary antibodies diluted in blocking buffer overnight at 4°C with gentle agitation
After wash 10min for 3 times, membranes were then incubated with HRP-conjugated secondary antibodies (1:5000) for 1 hour at room temperature.
After washing three times with TBST, membranes were developed using enhanced chemiluminescence (ECL) reagent.
Imaging
Signals were detected using ImageLab (ver. 5.2.1) (Biorad)