Matthieu Sanial, Ryan Miled, Marine Alves, Sandra Claret, Nicolas Joly, Véronique Proux-Gillardeaux, Anne Plessis, Sébastien Léon
{"title":"直接观察凝胶中的荧光蛋白:一种快速、经济、定量的免疫印迹替代方法","authors":"Matthieu Sanial, Ryan Miled, Marine Alves, Sandra Claret, Nicolas Joly, Véronique Proux-Gillardeaux, Anne Plessis, Sébastien Léon","doi":"10.1111/boc.202400161","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background Information</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Methods and Results</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Conclusions and Significance</h3>\n \n <p>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.</p>\n </section>\n </div>","PeriodicalId":8859,"journal":{"name":"Biology of the Cell","volume":"117 2","pages":""},"PeriodicalIF":2.4000,"publicationDate":"2025-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/boc.202400161","citationCount":"0","resultStr":"{\"title\":\"Direct observation of fluorescent proteins in gels: A rapid, cost-efficient, and quantitative alternative to immunoblotting\",\"authors\":\"Matthieu Sanial, Ryan Miled, Marine Alves, Sandra Claret, Nicolas Joly, Véronique Proux-Gillardeaux, Anne Plessis, Sébastien Léon\",\"doi\":\"10.1111/boc.202400161\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background Information</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods and Results</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions and Significance</h3>\\n \\n <p>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. 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Direct observation of fluorescent proteins in gels: A rapid, cost-efficient, and quantitative alternative to immunoblotting
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 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.
期刊介绍:
The journal publishes original research articles and reviews on all aspects of cellular, molecular and structural biology, developmental biology, cell physiology and evolution. It will publish articles or reviews contributing to the understanding of the elementary biochemical and biophysical principles of live matter organization from the molecular, cellular and tissues scales and organisms.
This includes contributions directed towards understanding biochemical and biophysical mechanisms, structure-function relationships with respect to basic cell and tissue functions, development, development/evolution relationship, morphogenesis, stem cell biology, cell biology of disease, plant cell biology, as well as contributions directed toward understanding integrated processes at the organelles, cell and tissue levels. Contributions using approaches such as high resolution imaging, live imaging, quantitative cell biology and integrated biology; as well as those using innovative genetic and epigenetic technologies, ex-vivo tissue engineering, cellular, tissue and integrated functional analysis, and quantitative biology and modeling to demonstrate original biological principles are encouraged.