Western Blotting with Solutions containing Nanoliter Volumes of Antibody.

Abstract

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. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. 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. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. Complementary diagrams, images, and videos are provided. The protocol is demonstrated using 0.3 nanoliters of anti-serum to detect fibronectin in a human foreskin fibroblast cell line. Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. Published 2019. This article is a US Government work and is in the public domain in the USA.