研究目的
To develop novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs for live-cell protein labeling with lipocalins.
研究成果
The study successfully developed novel red-shifted fluorogenic compounds for live-cell protein labeling with lipocalin-based protein hosts. The emission of the compound 3 in complex with the lipocalin DiB1 was considerably shifted to the red region of the spectrum, offering potential for lower phototoxicity in light-intense applications.
研究不足
The study focused on the synthesis and initial application of novel fluorogenic compounds. Further optimization and testing in various biological systems may be required to fully understand their potential and limitations.
1：Experimental Design and Method Selection
The study involved the synthesis of novel red-shifted fluorogenic GFP chromophore analogs and their application in live-cell protein labeling. The methodology included the synthesis of two groups of chromophores: conformationally locked by fluoroboric or oxygen bridge compounds and non-locked compounds.
2：Sample Selection and Data Sources
The study used commercially available reagents and solvents for synthesis. The optical properties of the synthesized compounds were determined using UV/VIS and fluorescence spectrophotometry.
3：List of Experimental Equipment and Materials
Equipment included a Varian Cary 100 UV/VIS Spectrophotometer, Varian Cary Eclipse Fluorescence spectrophotometer, and a Nikon Eclipse Ti N-STORM microscope for super-resolution imaging. Materials included various chemical reagents for synthesis and cell culture media for biological experiments.
4：Experimental Procedures and Operational Workflow
The synthesis involved condensation reactions, oxidation, and purification by column chromatography. The biological experiments included cell culture, transfection, and fluorescence microscopy.
5：Data Analysis Methods
Fluorescence titration data were fitted to a 1:1 binding model to determine dissociation constants (Kd). Super-resolution images were rendered with ThunderSTORM software.
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