Detection of protein expressed using cell-free systems is required for most applications such as protein:protein interaction and protein:nucleic acid interaction studies. Traditionally, one adds radioactive [35S]methionine to cell-free expression reactions, and the methionine is incorporated into the expressed protein, allowing detection by autoradiography. Many researchers are moving away from radioactivity. Traditional Western blot analysis provides the researcher a nonradioactive method for detection but, if performed improperly, can result in high background, which can mask expressed proteins and affect downstream applications.
One critical step in producing low-background, high-signal Western blots is choosing the correct dilution of the primary antibody. Typically the manufacturer recommends antibody dilution from 1:1,000 to 1:2,500 for standard western blotting experiments. However when using crude lysates as a source of the target protein, these recommendations exhibit significant background. When the antibody was diluted 1:50,000, background was decreased significantly, and the positive signal was a large percentage of the total signal.
As a general recommendation when performing Western blot analysis of proteins expressed in cell-free systems, one must experimentally determine the optimal dilution of the primary antibody. In the Western blots performed in this study, primary antibodies were diluted ~50-fold more than the provider’s recommended dilution.
For additional technical details refer to this recent article published in Promega’s PubHub:
Hook, B and Schagat, T. (2011) Non-Radioactive Detection of Proteins Expressed in Cell-Free Expression Systems Promega Corporation Web site. Accessed August 17, 2011.