Syracuse

Background Information: The discovery of green fluorescent protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility, and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. Methods and Results: In this study, we report that various green, orange, and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading, and migration on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the timeconsuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Conclusions and Significance: Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable to or better than antibody-based detection, a better quantification, and a broader dynamic