ArticleNature protocols2014
Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications.
Article in Nature protocols, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 27 papers.
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Who cites it
27 citing papers in PubMed, 60 citations in OpenAlex.
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- Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.Protein science : a publication of the Protein Society · 2024Article
- A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent Protein.Biochemistry · 2024Article
- Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.Biosensors · 2023Article
- Automating the High-Throughput Screening of Protein-Based Optical Indicators and Actuators.Biochemistry · 2023Review
- Single-molecule counting applied to the study of GPCR oligomerization.Biophysical journal · 2022Review
- Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification.Nature communications · 2022Article
- Rapid directed molecular evolution of fluorescent proteins in mammalian cells.Protein science : a publication of the Protein Society · 2022Article
- Cyan fluorescent proteins derived from mNeonGreen.Protein engineering, design & selection : PEDS · 2022Article
- Excitation ratiometric chloride sensing in a standalone yellow fluorescent protein is powered by the interplay between proton transfer and conformational reorganization.Chemical science · 2021Article
- Versatile phenotype-activated cell sorting.Science advances · 2020Article
- HriGFP Novel Flourescent Protein: Expression and Applications.Molecular biotechnology · 2020Article
- Tutorial: guidance for quantitative confocal microscopy.Nature protocols · 2020Review
- Quantitative Bio-Imaging Tools to Dissect the Interplay of Membrane and Cytoskeletal Actin Dynamics in Immune Cells.Frontiers in immunology · 2020Review
- A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.Nature chemical biology · 2018Article
- A genetically encoded CaBMC biology · 2018Article
- Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation.The journal of physical chemistry letters · 2017Article
- Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks.Methods in enzymology · 2017Article
- Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.PloS one · 2017Article
Corrections and comments
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Authors and funding
5 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.
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