![]() Predictably, specific and sensitive antibodies are a crucial component of successful western blotting assays. Though the nuances of a western blotting procedure vary with application, the basic steps of all workflows remain consistent. The separated proteins are transferred to, and immobilized on, a membrane and protein:antibody complexes are created to allow protein detection and analysis. Following denaturation, a protein sample is subjected to electrophoresis using a polyacrylamide gel capable of separating any two proteins of different apparent molecular masses ( Gallagher 2007). Western blotting developed as a means of reproducibly separating and analyzing proteins (Towbin, et al 1979). The desire to obtain a comprehensive understanding of a target’s transcription, translation, and expression has led to the development of numerous key protocols including southern blotting, northern blotting, western blotting, fluorescence in situ hybridization, and others ( Gallagher, et al, 2001 Wang, et al 2018). Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. The protocol is demonstrated using 0.3 nanoliters of anti-serum to detect fibronectin in a human foreskin fibroblast cell line. Complementary diagrams, images, and videos are provided. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. This article provides a step by step protocol for detecting proteins of interest with solutions containing nanoliter volumes of antibody without altering the preceding gel electrophoresis and transfer methods. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. ![]() Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol’s western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. ![]()
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