Western blot: technique, theory, and trouble shooting
Open accessAbstract
Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.
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The blot intensity was quantified using Image J software and the results were reported relative to tubulin band density used as a load control.
“The blot intensity was quantified using Image J software and the results were reported relative to tubulin band density used as a load control [28].”
From Intervention by picroside II on FFAs induced lipid accumulation and lipotoxicity in HepG2 cells (2021) · cites this paper as [28]
The Theory > Quantification section says western blots provide relative comparisons of protein levels and that differences in loading and transfer need to be standardized (page not available). It does not mention ImageJ or reporting results relative to tubulin band density, so it supports only the general rationale for relative quantification. (LLM, conf=0.84)
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- Authors
- Yang, P.-C., & Mahmood, T.
- Affiliations
- McMaster University (CA)
- Corresponding author
- Yang, Ping-Chang
- Venue
- North American Journal of Medical Sciences
- Published
- 2012
- Volume
- 4
- Issue
- 9
- Pages
- 429
- ISSN
- 1947-2714 (ISSN-L)
- PMC
- PMC3456489
- OpenAlex
- W2116240218
- Cited by
- 1,421
- References
- 0
- Citations by year
- 2026: 124 · 2025: 147 · 2024: 145 · 2023: 154 · 2022: 154 · 2021: 158 · 2020: 129 · 2019: 107 · 2018: 107 · 2017: 83 · 2016: 52 · 2015: 36 · 2014: 20 · 2013: 3 · 2012: 1
- FWCI
- 7.72
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- cc-by-nc-sa
- Locations
- North American Journal of Medical Sciences (publishedVersion)
- PubMed Central (publishedVersion, cc-by-nc-sa)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Keywords
- Western blot, protein detection, membrane transfer, troubleshooting, protein extraction
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