Fluorescent Protein Probes
Fluorescent Protein Probes - Related Videos
11:21
Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
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Cited by 5 •
2010
This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.
08:46
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
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Cited by 34 •
2015
A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.
12:52
Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II
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Cited by 4 •
2015
Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.
06:26
Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography
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Cited by 2 •
2023
The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.
04:02
An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells
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2025
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.
FAQs
The chromophore absorbs energy from illumination at an appropriate wavelength and then releases part of that energy as emitted light. This absorption and emission pattern determines when the probe can be detected by fluorescence microscopy. Consequently, the selected probe must match the illumination conditions and the biological question, such as observing a particular cell or tracking a molecular event.
Engineered variants can modify brightness, emitted color, stability, or sensitivity to cellular conditions. These changes allow researchers to select a probe suited to a specific imaging task rather than using one signal for every experiment. A brightness-adjusted variant may support clearer observation, while a condition-sensitive variant can report changes occurring inside cells.
A probe linked to a target protein helps reveal that protein’s location and movement, whereas a responsive probe changes its fluorescent behavior in relation to ions or other molecular changes. The first approach emphasizes spatial localization, while the second reports a biological condition or event. Together, these designs connect molecular behavior with broader cellular activity.
Illumination must provide a wavelength that the chromophore can absorb, because unsuitable excitation will not produce the intended fluorescent signal. Cellular conditions also matter when a probe is engineered for sensitivity to ions or other molecular changes. In those cases, the observed fluorescence can reflect both probe design and the condition being monitored in the cell.
A conceptual workflow begins by choosing a probe whose color, brightness, stability, or sensitivity fits the experiment. Researchers then genetically encode it in the relevant biological system, link it to a target when localization is needed, or use a responsive design for molecular changes. Fluorescence microscopy provides the illumination and records the resulting signal for analysis.
These probes can support studies of protein localization, gene expression, signaling activity, and dynamic processes in living cells. Their signals provide a way to observe molecular events without removing the cells from the imaging context. In biology, this helps researchers relate changes at the molecular level to cellular behavior and, where relevant, tissue-level patterns.