Fluorescent Modification

What is Fluorescent Modification?

The compounds on which fluorescence modification depends are called fluorescent substances. Most fluorescent substances are compounds with a conjugated double bond system chemical structure. When this type of compound is irradiated with ultraviolet light or blue-violet light, the compound absorbs energy to transition from the ground state to the excited state. The excited state electrons are unstable. When the ground state is restored from the excited state, fluorescence is emitted. Fluorescence modification technology is a technology that connects the covalent bonds of fluorescent groups to the molecules to be studied, such as peptides, proteins, and nucleic acids. Use the fluorescent properties of fluorescent substances to provide information about the object under study. Fluorescence modification technology has the advantages of no radioactivity, simple operation, high sensitivity and good selectivity, and is widely used in many research fields, especially biological research.

Choice of Fluorescent Substances

Choose the appropriate fluorescent substance according to the experimental needs.

For in vivo studies, fluorescent substances with an emission wavelength of 650-900 nm, such as ICG and Cy5.5, are generally selected. The fluorescence emitted by such substances has better tissue penetration ability and is less affected by background interference.

For in vitro studies, fluorescent substances with an emission wavelength of 400 to 600 nm, such as FITC and TAMRA, are generally selected.

Fluorescence Modification of Peptides

• Modification Site

Fluorescence modification of peptides is an important content in the field of peptide synthesis. Fluorescent modifications can be attached to the N-terminus or C-terminus of the peptide sequence by covalent bonds. The fluorescent substance can be directly connected to the N-terminus of the peptide, or it can be connected to the side chain of lysine (or cysteine) at the C-terminus of the peptide.

• Effect

Fluorescent substance-modified peptides can be used for in vivo imaging, protein binding and localization studies. Fluorescently modified peptides combined with imaging techniques can be used to identify specific targets. In vitro imaging by confocal or fluorescence microscopy is one of the most effective methods to study various biological processes and various interactions within cells. Fluorescently modified peptides localize to specific targets on actin and do not easily aggregate proteins, making them ideal for in vitro tracking. In addition, fluorescently modified peptides can be used to detect the activity of target proteins. Fluorescence modification can be used not only for the tracking of polypeptides in living cells, but also for the study of enzymes. Fluorescence resonance energy transfer (FRET) is a mechanism that describes the energy transfer between two fluorophores. When applying FRET, the substrate peptide often contains a fluorescent labeling group and a fluorescence quenching group. When the peptide remains intact, the fluorescent quenching group quenches the fluorescent labeling group and no fluorescence is detected. The labeling group fluoresces when the peptide sequence is cleaved by protease activity. This fluorescence can be continuously detected, allowing quantitative analysis of enzyme activity. FRET peptides are suitable substrates for a variety of enzymatic studies.

In the fluorescence modification technology, the most commonly used fluorescent substances are FITC, FAM, TAMRA, etc., which can be used for the modification of proteins and peptides.

• FITC Modification

FITC is an amine-reactive fluorescent dye that can be used to modify peptides. FITC typically reacts with the N-terminal amino group of the peptide. During N-terminal labeling, an alkyl spacer, such as aminocaproic acid (Ahx), is introduced between the amino group and the thiourea bond formed by the reaction of FITC. In this way, the peptide and FITC can be coupled without hindrance. FITC-labeled cell penetrating peptides (CPPs) can be used to image intracellular components.

• FAM Modification

FAM-modified peptides are commonly used in confocal microscopy and flow cytometry applications.

• TAMRA Modification

TAMRA is one of the most commonly used orange fluorescent substances for labeling peptides and proteins. TAMRA has several advantages over FITC. For example, TAMRA showed stronger resistance to photobleaching than FITC. Furthermore, TAMRA has an additional positive charge that may increase the interaction between TAMRA and target compounds. FITC and TAMRA can be used in combination. Because they have different excitation and emission wavelengths and do not quench each other.

• Coumarin Modification

Coumarin-modified peptides are commonly used in the trace determination of enzymes, the identification of enzymes, and the preparation of laser dyes.

• ATTO Modification

ATTO dyes offer better photostability and ozone resistance, longer signal lifetime, and lower background for increased sensitivity. They are ideal for composite technologies using visible and near-infrared emission wavelengths.

• Dinitrophenyl (Dnp) Modification

Dnp is a non-fluorescent dye that can be used as a fluorophore quencher.

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