Rapid, Antibody-Free Detection of Recombinant Proteins on Blots Using Enzyme Fragment Complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this chapter, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of posttranslational protein modifications such as glycosylation.

Measurements of ß-arrestin recruitment to activated seven transmembrane receptors using enzyme complementation.

The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an emerging protein-protein interaction technology based on the forced complementation of a split enzyme that has proven to be highly effective in monitoring the formation of GPCR-arrestin complexes. In these systems, the target proteins are fused to two fragments of an enzyme that show little or no spontaneous complementation. Interaction of the two proteins forces the complementation of the enzyme, resulting in an enzymatic measure of the protein interaction. This chapter discusses the utility and methods involved in using the PathHunter ß-galactosidase complementation system to monitor arrestin recruitment and the advantages of exploiting this pathway in the characterization of 7TMR function.

Rapid, antibody-free detection of recombinant proteins on blots using enzyme fragment complementation.

Alternative, antibody-free techniques to western analysis of protein blots can offer reduced assay times for routine analysis of expression of recombinant proteins. We have adapted the commercially available enzyme fragment complementation technology to provide a rapid protein detection method for protein blots based on significantly reducing the number of incubation and washing steps used in traditional approaches, and eliminating the requirement for antibodies. In this article, we highlight the use of this assay for measuring recombinant protein expressed in mammalian cells for a range of applications, including dot blot screening of large numbers of different cell samples, assessment of protein integrity through detection of degradation bands, and characterization of post-translational protein modifications such as glycosylation.