Feb 13, 2026

Public workspaceWestern blot

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwn2k7vmk/v1
  • Guangli Suo1
  • 1CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu, 215123, China.
  • MTS
Guangli Suo
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwn2k7vmk/v1
Protocol Citation: Guangli Suo 2026. Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwn2k7vmk/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: February 13, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 243186
Keywords: western blot the protein level, brc tissue, western blot, protein level, mtss
Abstract
The protein levels of BRC tissue and MTSs was detected via western blot.
Materials
- Cold RIPA lysis buffer (Beyotime)
- BCA protein assay kit (Beyotime)
- 10% SDS-PAGE gels
- Polyvinylidene difluoride (PVDF) membranes (Merck)
- 5% skim milk
- HRP-conjugated secondary antibodies
- Fujifilm LAS 4000 luminescent image analyzer (Fujifilm Life Science)
- Quantity One software (Bio–Rad)
- Primary antibodies against PARP1, Bax, Actin, NF-κB, p-NF-κB, P38, p-P38, β-Catenin, full-length and cleaved caspase-3 (CST)
- Primary antibodies against GAPDH, Notch1, CD44, CD24, ALDH1A1, P53, PCNA, c-Myc, YAP (Proteintech Company)
- Goat anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Beyotime Company)
Troubleshooting
Western blot
Harvest and lyse tissues or sorted MTSs from MWC-chips in cold RIPA lysis buffer (Beyotime).
Centrifuge at 12,000 rpm for 15 min at 4°C.
Clarify lysates and collect supernatants for protein concentration determination using the BCA protein assay kit (Beyotime).
Fractionate proteins on 10% SDS-PAGE gels and transfer to polyvinylidene difluoride (PVDF) membranes (Merck).
Block PVDF membranes with 5% skim milk at room temperature for 1 hour.
Incubate overnight with primary antibodies.
Apply HRP-conjugated secondary antibodies to membranes for an additional hour.
Visualize blots using chemiluminescence and photograph using Fujifilm LAS 4000 luminescent image analyzer (Fujifilm Life Science).
Quantify grayscale values using Quantity One software (Bio–Rad).