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Protein peptide analysis tool for peptide hydrophilicity

LifeTein Fluorescent Modifications: FITC, FAM, TAMRA, Biotin, EDANS/Dabcyl, and more

Fluorescent dyes are the primary means of labeling biomolecules. Fluorescein derivatives are probably the most widely used. They have a wide range of applications in fluorescence microscopy, flow cytometry, and immunofluorescence-based assays.

FRET Peptide synthesis: FRET Assay

  • FRET Assay
  • FAQs
  • Case Studies

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the excited states of two dye molecules. Excitation is transferred from a donor molecule to an acceptor molecule without the emission of a photon. This assay is quick, extremely sensitive, and uncomplicated to perform.

FRET can be performed using just one type of dye, but most applications usually call for two. FRET describes the transfer of energy from an initially excited donor (dye 1) to an acceptor (dye 2). Typically, this donor emits light at a wavelength λd. It overlaps with acceptor's absorption wavelength λa. If donor and acceptor dye are close at 10 – 100 Å, the energy transfer begins in one of two ways below:

  • a) The transferred energy may be converted to molecular vibrations (if the acceptor is a dark quencher)
  • b) The transferred energy may be emitted as light with a longer wavelength (if the acceptor is fluorescent).

FRET Overview: EDANS and Dabcyl

A donating group (EDANS) and accepting group (DABCYL) are attached to a substrate of HIV protease. If the substrate is not cleaved, DABCYL will quenche EDANS and fluorescence cannot be detected. After HIV-1 protease cleaves the substrate, EDANS is not quenched by DABCYL. The EDANS fluorescence can then be detected. Using changes in the intensity of EDANS fluorescence, the technique can be used to screen the effectiveness of the protease inhibitor.

FRET Peptide synthesis: EDANS and Dabcyl

FRET peptides are useful for the study of peptidase specificity. This is a rapid screening method to determine enzymatic activity based on the continuous monitoring. The activity of nanomolar concentrations of the enzyme can be measured efficiently by the hydrolysis reactions of peptide bonds between the donor and acceptor pair. The FRET peptides quench internal fluorescence when there is no activities. When the peptide bond between the donor and acceptor pair is cleaved, the fluorescence is activated continuously. Then the enzyme activity are measured at a quantitative level.

FRET peptides are used as substrates in enzyme studies:

  1. Kinetic and functional characterization of peptidases, kinases, proteases, and phosphatases.
  2. Screening, monitoring and detection new proteolytic enzymes.
  3. Conformational investigation of peptide folding.

Standard dye combinations used for FRET:

  1. Fluorescein and Dabcyl:FAM/Lys(Dabcyl)
  2. Fluorescein and Tamra: FAM/TAMRA
  3. Methoxy-coumarin-acetic-acid(MCA) and 2,4-Dinitrophenyl(DNP): MCA/Lys(Dnp).
  4. Ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) or N-(2,4-dinitrophenyl)ethylenediamine (EDDnp): Abz/Tyr (NO2), Abz/EDDnp
  5. Dabcyl and Glu(EDANS)

Donor-acceptor pairs capable of quenching using resonance energy transfer in peptide substrates of proteolytic enzymes


Wavelengths (nm)





Tyr (NO2)




Forster Critical Distance for Common RET Donor-Acceptor Pairs



Forster Distance





FITC Peptide synthesis: FITC modification

The fluorescein isothiocyanate (FITC) is more reactive than carboxy fluorescein, which must also be activated before use. FITC can react extensively with sulfhydryl groups such as the side chains of reduced cysteine residues.

FITC Peptide synthesis: FITC labeling process

For many applications, fluorescent labels can be introduced during chemical synthesis. After selective unmasking of protecting group, FITC may react with either lysine, or with a primary amino group at the N terminus of the growing peptide. An alkyl spacer called aminohexanoic acid (Ahx) is introduced between the last amino acid and the thiourea linkage generated through the reaction of isothiocyanate and amine.

At the acidic conditions for cleavage, N-terminal FITC-labeled peptides undergo a cyclization. This causes a removal of the last amino acid. The situation can be avoided when an amino hexanoic acid spacer is used.

The steric hindrance consideration is the major reason to use the Ahx. Ahx or b-Ala have been used successfully as spacers in the generation of FITC-labeled peptides.

FITC Peptide synthesis: FITC

Tetramethylrhodamine (TAMRA) is another reactive fluorophore that can exhibit suboptimal properties for intracellular reactions. TAMRA-based probes have the most uniform intracellular distribution and best cytosolic diffusivity. This TAMRA chromophore offers several advantages over fluorescein. TAMRA is more resistant to photobleaching compared to fluorescein. Fluorescein is typically excited at 494 nm and emits at 520 nm. In contrast, TAMRA's absorbance and fluorescence maxima is at 546 nm and 580 nm, respectively. In comparison with fluorescein, TAMRA has one extra positive charge. This could increase the interaction between TAMRA and the target compound and helps improve binding of the probe to the compound. Similar to Cy3, TAMRA is an excellent flourochrome for its stability and brightness. There is no problem in combination of FITC and TAMRA because their excitation and emission maxima are well separated and they do not quench each other.

Usually, dyes such as biotin and FITC can be introduced at either the N-terminal or C-terminal. We recommend modifications at the N terminus because it is more likely to succeed and has a shorter turnaround time. This is because peptides are synthesized from the C terminus to the N terminus, making N terminal modification the last step in the SPPS protocol. Unlike modification at the C terminus, it requires no additional coupling steps.

Most dyes are large aromatic molecules, and the incorporation of these bulky molecules can prevent interactions between the label and the peptide. This helps to maintain the peptide conformation and its biological activity. We recommended the inclusion of a flexible spacer, such as the six-carbon linker Ahx, in synthetic peptide projects. This renders the fluorescent label more stable. FITC can also be easily linked to a cysteine thiol moiety or to the amino group of lysine at any position.

Probe Ex (nm) Em (nm) MW Notes
Methoxycoumarin (MCA) 360 410 317 Succinimidyl ester
Fluorescein (FITC) 495 519 389 pH sensitive
X-Rhodamine 570 576 548
Rhodamine B 570 590

Click to see our comprehensive list of fluorophores including absorption and emission peak values.

Frequently Asked Questions: Peptide Synthesis

Please click here to see more FAQs

Is a spacer required for fluorescent modification?

How should I dissolve peptides?

How do I choose the best level of peptide purity for my research?

Case Study 1

This case study shows the approach used in most FRET assays. The fluorescent molecule methoxycoumarine acetic acid (MCA) is incorporated at the N-terminus of the peptide. The peptide in question was designed to serve as a substrate for the matrix metalloprotease stromelysin. The quencher N-3-(2, 4-dinitrophenyl)-L-2,3-diaminopropionyl (DPA) was incorporated after the scissile Gly-Leu peptide bond had been hydrolyzed by the enzyme (Knight el al., 1992).

The complete peptide sequence is:


Peak MCA absorbance takes place at 328 nm and peak fluorescence at 393nm. The DPA group has a strong absorption band at 363 nm and a prominent shoulder at 410 nm. This shoulder overlaps with the fluorescence band of MCA and can quench fluorescence.

A 1 μm solution of MCA-Pro-Leu (the product of enzymatic hydrolysis) was found to be 130 times more fluorescent than a comparable solution of the MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2, showing excitation and emission at 328 nm and 393 nm, respectively (Knight el al., 1992).

Enzymatic hydrolysis of this peptide cause separation of the MCA and DPA groups and a large increase in MCA fluorescence. This increase in fluorescence can serve as an indicator of the speed of the reaction. This has allowed investigators to establish the values of Kcat/Km of this substrate for several members of the matrix metalloprotease family. This assay was recently used to determine the potency of potential inhibitors of stromelysin through measurement of the effects of the inhibitors on the initial velocity of the enzymatic reaction (Copeland et al., 1995)

Case Study 2

The client requested a very hydrophobic peptide 68 amino acid in length (85% purity) with FITC modification at the N-terminus. The peptide was successfully synthesized in 4 weeks.

HPLC Results:

Peptide synthesis: FITC modification HPLC

MS Results:

Peptide synthesis: FITC modification MS


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