Fluorescent Modifications for Peptides
GenScript offers numerous fluorescent tags for peptides,
and our repertoire is being expanded continuously.
Listed below are a few of our most commonly used
modifications:
| Name |
Excitation (nm) |
Emission (nm) |
Emission color |
Application Fields |
| 7-Methoxycoumarin-4-acetic acid |
328 |
393 |
Blue |
In vitro imaging Subcellular
localization Confocal microscopy Flow
cytometry
|
| FITC-Ahx |
494 |
521 |
Green |
| FAM |
495 |
520 |
Green |
| Cy3 |
555 |
570 |
Yellow |
| 5-Carboxytetramethylrhodamine (TMR) |
542 |
568 |
Orange |
| Cy5 |
646 |
662 |
Red |
| Cy5.5 |
673 |
707 |
Near-infrared |
In vitro imaging Subcellular
localization In vivo optical imaging
Angiography
|
| Cy7 |
750 |
773 |
Near-infrared |
FRET pairs
Fluorescence resonance energy transfer (FRET) is a
mechanism that describes the energy transfer between two
fluorophores. Since FRET efficiency is partly based on
distance between a donor and acceptor molecule, this
technique is commonly used for studying enzyme
efficiency, protein-protein interactions, or other
molecular dynamics (Fig 1).
Fig 1. FRET mechanism for protease studies. When
the peptide remains intact, the acceptor molecule will
quench the donor molecule, and no fluorescence will be
detected. If the sequence is cleaved by protease
activity, the acceptor will no longer quench the donor,
and a fluorescent signal will be detected.
Below are the most commonly used FRET pairs:
| Donor |
Acceptor |
|
|
|
|
| Name |
Excitation (nm) |
Emission (nm) |
Name |
Excitation (nm) |
Emission (nm) |
| Cy2 |
490 |
510 |
Cy3 |
555 |
570 |
| FITC |
494 |
521 |
TRITC |
557 |
576 |
| FAM |
495 |
520 |
Cy3 |
555 |
570 |
| FAM |
495 |
520 |
Texas Red |
589 |
615 |
| FAM |
495 |
520 |
Cy5 |
646 |
662 |
| Cy3 |
555 |
570 |
Cy5 |
646 |
662 |
| EDANS |
335 |
493 |
DABCYL |
453 |
- |
| Glu(EDANS)-NH2 |
335 |
493 |
DABCYL |
453 |
- |
| MCA |
328 |
393 |
DNP |
348 |
- |
| Abz |
330 |
420 |
DNP |
348 |
- |
| Abz |
330 |
420 |
Tyr (3-NO2) |
360 |
- |
Fluorescent peptides for subcellular imaging
In vitro imaging by confocal or fluorescent microscopy
remains one of the most efficient and effective methods
for studying a variety of biological process and
interactions in cells. When combined with imaging,
fluorescent-labelled peptides can be used to identify
specific targets.
Read More »
For example, cell penetrating peptides (CPPs) modified with FITC and photoactivatable probes have been used to track binding patterns and dynamic behavior over time. Unlike proteins, these peptides localize to specific targets on actin and are less prone to protein aggregation, making them ideal for in vitro tracking (Pan 2014). Similarly, FITC-labeled CPPs have been used to image intracellular compartments of cells with low risk of cytotoxicity (Kirkham 2015).
Fluorescent peptides for angiography
In vivo imaging for angiography continues to be a major
application for fluorescent-labeled peptides. This
technique, in which contrast agents are used to image
the inside of blood vessels, enables physicians to
choose the most appropriate medical strategies or
treatments for an individual.
Read More »
For example, high resolution near-infrared fluorophores have been used as fibrin imaging-agents for deep vein thrombosis (DVT). Using Cy7 labeled fibrin-targeting peptides, CT scanning and confocal microscopy were used to differentiate between acute and subacute murine DVT (Hara 2012). Similar techniques have also been used for detecting apoptosis as a symptom for glaucoma. Fluorophore-labeled peptides that are activated by caspases can be imaged in vivo to track glaucoma progression (Qiu 2014).
FRET for screening proteolytic peptides
Proteolytic enzymes play important roles in infectious
diseases, which makes them a target for developing novel
therapeutics. To identify the peptide sequences that
these enzymes target, peptide libraries are often used.
The potential protease target sequences are combined
with FRET pairs, such that when the enzyme cleaves the
target peptide, a fluorescent signal can be detected.
Read More »
In these studies, a donor molecule, such as Abz (Marcondes 2015) or Lucifer Yellow (Rossé 2000) is covalently attached to the C-terminus, while acceptor molecules (ex. Dabsyl, DNP) are coupled to the N-terminus. This strategy is commonly employed with peptides synthesized using a solid-phase approach, and peptides are left conjugated to the bead. If the peptides are not targeted by protease activity, both the donor and acceptor will be present, and the bead will appear non-fluorescent. If the peptides are cleaved, the peptide will no longer be quenched by FRET and the beads will fluoresce. Since protease studies are most effective when FRET is combined with shorter peptides, micro-scale peptide libraries are ideal choices for these experiments.
References
Fluorescent Modification Case studies
Fluorescent peptide labels have numerous research
applications, and GenScript has extensive experience
synthesizing peptides with a variety of modifications.
Case Study 1
Sequence: LYRLGLGH
Modification: MCA/DNP
Quantity: 1-4 mg
| Required purity |
Estimated Turnaround time |
Actual purity |
Actual turnaround time |
>98%
|
17 days |
99.563%
|
14 days |
Case Study 2
Sequence: IKDLSKEERLWEVQRILTALKRKLREA
Modification: 5-FAM (N-terminal)
Quantity: 10-14 mg
| Required purity |
Estimated Turnaround time |
Actual purity |
Actual turnaround time |
| >98% |
23 days |
99.10% |
13 days |
Case Study 3
Sequence: RAKWNNTLKQIASK
Modification: FITC-Ahx (N-terminal)
Quantity: 5-9 mg
| Required purity |
Estimated Turnaround time |
Actual purity |
Actual turnaround time |
| >98% |
17 days |
99.81% |
5 days |
