Fluorescent tag

Fluorescent labeling is a method of Biochemistry for labeling biomolecules with fluorophores.

Principle

Biomolecules are occasionally provided to facilitate tracing with fluorescent molecules. Through the coupling of fluorophores to proteins or carbohydrates on the cell surface, cells can be labeled directly or indirectly be detected with fluorescent immunoconjugates, eg by flow cytometry, fluorescence microscopy or fluorescence tomography.

When a fluorescent label the molecule to be detected can be directly provided with a fluorophore or detected indirectly by selectively binding fluorescent molecules. Methods for fluorescent labeling are:

  • Direct molecular evidence chemical coupling (only in vitro)
  • Bio-orthogonal labeling
  • Fluorescent reporter molecules
  • Fluorescent protein tags, protein tags with Fluorophorkopplung (Flash - day ) or RNA tags
  • Immunolabeling
  • Hybridization probes
  • Biotinylation

Proteins

Proteins at the green fluorescent protein (GFP ) can be used as a recombinant fusion protein or as a reporter gene in vivo, among other things. Inteins can be used to C-terminal labeling of proteins. Also the flash day may be used as fluorescent protein tag.

Proteins can also be semi-synthetic ( bioorthogonal ) are selected in which reactive amino acid derivatives in vivo are incorporated, which are coupled in the connection in vitro with a fluorophore. Furthermore, proteins with different chemical coupling reactions in vitro are provided with different fluorophores. For example, nucleophilic groups ( amino groups are most common in proteins ) by isothiocyanates with different fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and their derivatives are selected. Proteins can also be detected indirectly by immunolabeling with fluorescent immunoconjugates, such as fluorescence microscopy, the Western blot and immunoassay. In the course of peptide synthesis, peptides can be labeled by using fluorescent amino acid derivatives. Through a Förster resonance energy transfer ( FRET), bioluminescence resonance energy transfer ( BRET ), fluorescence correlation spectroscopy (FCS ) or a bimolecular fluorescence complementation ( BiFC ) can be detected with fluorophores the neighborhood of two proteins.

Nucleic acids

DNA can be labeled bioorthogonal. This DNA can be in vivo or in vitro labeled nucleoside analogues that can be subsequently coupled with a fluorophore, for example, via the Staudinger reaction. DNA can be chemically provided with fluorophores. Oligonucleotides can be labeled by a phosphoramidite synthesis with fluorophores which are used for example in the QPCR, DNA sequencing and in situ hybridization. In addition, DNA can be enzymatically generated in the course of a polymerase chain reaction with fluorescent nucleotides or labeled with a ligase and a terminal deoxynucleotidyl transferase. DNA can be detected indirectly by biotinylation and fluorescent avidin. Among other things, fluorescein, fluorescent lanthanides, gold nanoparticles, carbon nanotubes or quantum dots are used for couplings as fluorophores.

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