Western Blotting a review: Principle, protocol and problem solving

Western blotting is a fundamental technique widely used in molecular biology and biochemistry to quantify protein expression levels modifications various biological samples. This study presents detailed protocol for performing WB, focusing on the principles steps involved process. The article discusses isolation of samples from cultured cells their quantification using BCA assay. importance emphasized ensure accurate loading proteins gel wells. A step-by-step guide provided electrophoresis, highlighting preparation resolving stacking gels migration under an applied voltage. Furthermore, procedure detailed, covering membrane selection electrophoretic transfer use blocking agents prevent non-specific antibody binding explained, along with incubation primary secondary antibodies. emphasizes significance negative positive controls validate experimental results. Lastly, common troubleshooting strategies are address potential issues encountered during process, such as absent or weak bands, unusual patterns, high background. In conclusion, this comprehensive offers researchers reliable optimized method analyse accurately, contributing advancement research.