Western Blotting a review: Principle, protocol and problem solving
Senior Laboratorian at Sankara Nethralaya Referral Laboratory, Medical Research Foundation, Chennai, India.
Research Article
GSC Advanced Research and Reviews, 2023, 16(03), 178–187.
Article DOI: 10.30574/gscarr.2023.16.3.0324
Publication history:
Received on 27 July 2023; revised on 15 September 2023; accepted on 18 September 2023
Abstract:
Western blotting is a fundamental technique widely used in molecular biology and biochemistry to quantify protein expression levels and modifications in various biological samples. This study presents a detailed protocol for performing WB, focusing on the principles and steps involved in the process. The article discusses the isolation of protein samples from cultured cells and their quantification using the BCA protein assay. The importance of protein quantification is emphasized to ensure accurate loading of proteins in gel wells. A step-by-step guide is provided for gel electrophoresis, highlighting the preparation of resolving and stacking gels and the migration of proteins under an applied voltage. Furthermore, the blotting procedure is detailed, covering membrane selection and the electrophoretic transfer process. The use of blocking agents to prevent non-specific antibody binding is explained, along with the incubation of primary and secondary antibodies. The article emphasizes the significance of negative and positive controls to validate the experimental results. Lastly, common troubleshooting strategies are provided to address potential issues encountered during the Western blotting process, such as absent or weak bands, unusual patterns, and high background. In conclusion, this comprehensive Western blotting protocol offers researchers a reliable and optimized method to analyse protein expression and modifications accurately, contributing to the advancement of molecular biology and biological research.
Keywords:
Sample preparation for western blot; Protein quantification; Gel electrophoresis; Western blot
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Copyright © 2023 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0
