The method allows scientists and clinicians to perform standard western blotting techniques with solutions containing nano-scale quantities of antibody. This protocol uses minimal quantities of primary antibody to detect proteins immobilized on a PVDF or nitrocellulose membrane. Support Protocol 2 uses 2% SDS in 10 mM sodium phosphate buffer to extract proteins and immediately proceeds to gel electrophoresis to minimize sample proteolysis. Support Protocol 1 employs either radioimmunoprecipitation assay (RIPA) lysis buffer or 2% sodium dodecyl sulfate (SDS) in 10 mM sodium phosphate buffer to sequentially isolate proteins and determine protein concentration of the acquired sample prior to electrophoresis. ![]() Support Protocols 1 and 2 describe two methods of extracting protein from a tissue culture sample using two different lysis buffers. Basic Protocol 1 describes a western blotting workflow using two different style miniature incubation chambers. This unit includes a set of protocols for analyzing protein samples via western blot using sub-microliter volumes of primary antibody. Whether screening small mammal serum during monoclonal antibody production or attempting to preserve a stock of precious antibody, a method to probe a transferred membrane with minimal primary antibody is a valuable tool. Targeting a single epitope allows for greater specificity and decreases the possibility of off-target labeling and resultant misguided analysis. In comparison, monoclonal antibodies bind selectively to a single epitope approximately 15 amino acids in length ( Lai, et al 2017). Unfortunately, the nature of polyclonal primary antibodies poses the potential for off-target tagging ( Lai, et al 2017). ![]() Most western blotting workflows utilize a non-conjugated primary antibody specific to the protein of interest followed by a species-specific labeled secondary antibody targeting the constant region of the primary antibody. ![]() 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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