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Abstract Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here we describe an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels. For this, the highly specific protein ligase Connectase is used to selectively fuse fluorophores to target proteins carrying a recognition sequence, the CnTag. Compared to Western blots, this procedure is faster, more sensitive, offers a better signal-to-noise ratio, requires no optimization for different samples, allows more reproducible and accurate quantifications, and uses freely available reagents. With these advantages, this method represents a promising alternative to the state of the art and may facilitate studies on recombinant proteins.