Western Blot Protocol
Principle
Western blotting (WB), a widely adopted analytical method, is employed to detect specific target proteins within complex samples using antigen-specific antibodies. This technique serves the purpose of identifying individual proteins and allows for semi-quantitative analysis. It encompasses three fundamental processes: size-based separation through gel electrophoresis, subsequent transfer to a solid support via blotting, and visualization of the target protein through probing.
Gel electrophoresis is utilized to separate sample proteins based on their polypeptide lengths. These separated proteins are subsequently transferred onto a nitrocellulose (NC) membrane, where they are subjected to probing with a primary antibody specific to the target protein antigen. Typically, the membrane is incubated with an antibody against the antigen of interest, followed by a secondary antibody, leading to the eventual detection of the target protein.
Material
| Reagents | Instruments and Consumables |
| Loading Buffer | Eppendorf Tube |
| Running Buffer | Eppendoff |
| Transfer Buffer | Centrifuge |
| Washing Buffer (PBST) | Incubation Plate |
| Blocking Buffer | Oscillating Table |
| Chemiluminescent Substrate | Electrophresis Apparatus |
| Protein Maker | Transfer Tank |
| Primary Antibody | Imager |
| Secondary Antibody-HRP |
Procedure
Fig. 1 The workflow of WB.
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Application
Fig. 2 In vivo GSDME-mediated pyroptosis.1
The WB analysis of mouse heart tissues involved the assessment of the relative expression levels of full-length GSDMD (GSDMD-F), GSDMD-N terminal (GSDMD-N), full-length GSDME (GSDME-F), GSDME-N terminal (GSDME-N), caspase-3, and cleaved-caspase-3. The expression of GSDMD remained at a low level without the cleavage of its N-terminal fragment. In contrast, mice treated with Antitumor antibiotics exhibited a substantial increase in both GSDME expression and the presence of its N-terminal fragment. Furthermore, a significant elevation in cleaved-caspase-3 expression was evident in Antitumor antibiotics-treated mice. These findings indicate the involvement of GSDME-mediated pyroptosis in the development of Antitumor antibiotics-induced cardiotoxicity.
Fig. 3 BC069792 regulates KCNQ4 as a downstream target gene.2
This study investigated the function of long non-coding RNA BC069792 in breast cancer: Conducting in vitro and in vivo functional experiments utilizing cell culture and murine models. Protein expression assessed via WB and immunohistochemical staining reveals a significant increase in KCNQ4 protein levels in the BC069792 overexpression group compared to the control group.
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References
- Zheng, Xinbin, et al. "Bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME." Life sciences 242 (2020): 117186.
- Zhang, Yunxiang, et al. "LncRNA-BC069792 suppresses tumor progression by targeting KCNQ4 in breast cancer." Molecular cancer 22.1 (2023): 1-19.
