FRET stands for fluorescence resonance energy transfer, or Förster resonance energy transfer. FRET is a mechanism describing excited state energy transfer from the initially excited donor to an acceptor. The donor molecules typically emit at shorter wavelengths that overlap with the absorption of acceptors. The process is a distance-dependent interaction between the electronic excited states of two molecules without emission of a photon.
FRET can be used for conformational investigation of peptide folding. FRET peptides are widely used as suitable substrates in enzyme studies, such as:
- Functional characterization of peptidases / proteases / kinases / phosphatases
- Kinetic characterization of peptidases / proteases / kinases / phosphatases
- Screening and detection of new proteolytic enzyme
Chromophore/Fluorophore – Quencher Pairs
|Chromophore/Fluorophore||Lambda max (nm)||Lambda em (nm)||Quencher|
How does a quencher work?
A quencher is very efficient at absorbing certain wavelengths. When near a dye that emits at the absorbed wavelength, the light is “quenched”, and no longer visible. Quenchers are very similar in structure to dyes. The difference is that they emit in undetectable ranges, or in undetectable amounts. The ability to quench is a function of distance from the dye in most cases. Molecular beacons are effective in that the quencher actually comes in contact with the dye. Different quenchers are best for different dyes.
Can a dye be conjugated to protein or peptide internally?
Yes. Use the succinimidyl ester version of the dye and conjugate it to either a N-terminal and Lysine side-chain amino linker. Other options include using a Lys-FAM or Lys-TAMRA for Solid Phase Peptide Synthesis. These dyes are already conjugated to the Lys side-chain amino linker.
What is the difference between a 5-FAM and FITC labeled protein and peptide?
Both 5-FAM (5-carboxyfluorescein, single isomer) and FITC (fluorescein-5-isothiocyanate, isomers) are forms of the fluorescent dye fluorescein. FITC refers to a particular form of reactive species, the isothiocyanate, of the dye. It yields a urea linkage upon reaction with a primary amine. 5-FAM is the preferred reagent for labeling a protein or peptide. It results in an amide bond when reacted with a primary amine. The chemistry is more robust and better yielding. Furthermore, it has been shown that 5-FAM is less susceptible to photo-bleaching. The 5-FAM and FITC conjugated protein and peptide have similar spectral properties.
Can I use a dye or modification that is not listed here?
Yes. Tell us the dye or modification you are looking to use; we will find out if it is commercially available. If it is not, there is a possibility we can make it in-house. If neither of these is an option we will do our best to see if there is another dye or modification that can give you similar results. If any of these options work for you, we will then send you a quotation.
What are the absorption max and emission max values for my fluorescent labeled protein or peptide?
Spectra data can be found (click here). These values are provided by the manufacturer of the fluorescent dye and are generally calculated from the free dye not attached to a protein or peptide. As a general rule, these values work fine for the common protein or peptide user as there is little change, if any at all, when the dye is attached to a protein or peptide. The particular base composition of a protein or peptide can play a role as can pH in many cases, particular fluorescein and related dyes.
Can you make a peptide with two different dyes or other conjugate?
The answer is complicated in that there are many ways to place different conjugations on a peptide, but we are still limited by what is available to us for conjugation and compatibility issues. The easiest way is if one or both of the conjugates are available as thiol of cysteine and/or support bound reagents. If at least one reagent is available, the other conjugate can often be attached through a primary amine at the other end of the peptide. Chemical compatibility is avoided in this case. If neither conjugate is available as a thiol of cysteine or support bound reagent, then we often will use an orthogonal linker scheme incorporating both a thiol linker and an amino linker. This requires that one of the conjugates is available as a maleimide, which is thiol specific under neutral pH conditions. Most of all the commonly used dyes are available in both the succinimidyl and maleimidyl ester forms. The most common, such as fluorescein, TET, HEX, TAMRA, Cy™5 and Cy3 are also available as primary amine or support bound reagents. We will help you design your specific peptide when you place your order. Also, see our Products section on dyes for more information on what is available.