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 time-consuming 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 range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.
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 range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.