| Title: |
Direct observation of fluorescent proteins in gels: A rapid, cost‐efficient, and quantitative alternative to immunoblotting |
| Authors: |
Sanial, Matthieu; Miled, Ryan; Alves, Marine; Claret, Sandra; Joly, Nicolas; Proux‐gillardeaux, Véronique; Plessis, Anne; Léon, Sébastien |
| Contributors: |
Institut Jacques Monod (IJM (UMR_7592)); Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité); ARCPJA2022050005002; ANR-18-IDEX-0001,Université de Paris,Université de Paris(2018); ANR-23-CE13-0012,AMPKill,Régulation de l'inhibition de la kinase AMPK par le métabolisme via les protéines 14-3-3(2023) |
| Source: |
ISSN: 0248-4900. |
| Publisher Information: |
CCSD; Wiley |
| Publication Year: |
2025 |
| Subject Terms: |
biochemistry; fluorescence techniques; yeast; GFP; Gel imaging; technique; [SDV]Life Sciences [q-bio]; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry; Molecular Biology/Biochemistry [q-bio.BM] |
| Description: |
International audience ; 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 |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
info:eu-repo/semantics/altIdentifier/pmid/39924827; PUBMED: 39924827 |
| DOI: |
10.1111/boc.202400161 |
| Availability: |
https://hal.science/hal-04956250; https://hal.science/hal-04956250v1/document; https://hal.science/hal-04956250v1/file/BOC-117-e2400161.pdf; https://doi.org/10.1111/boc.202400161 |
| Rights: |
http://creativecommons.org/licenses/by-nc-nd/ ; info:eu-repo/semantics/OpenAccess |
| Accession Number: |
edsbas.7782815C |
| Database: |
BASE |