Fluorescence Resonance Energy Transfer (FRET)
FRET is a process where energy from a luminescent molecule (the donor) that is in its electronically excited state is transferred to an acceptor molecule by dipole-dipole interaction. FRET occurs when donor and acceptor molecules are in close proximity (usually within 10 to 80 Angstroms). The acceptor molecule can either be another fluorescent dye or a non-fluorescent molecule. There are several parameters that play an important role for FRET to occur:
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| 1) |
The absorption spectrum of the acceptor molecule must overlap the emission spectrum of the donor characterized by the overlap integral (J). |
| 2) |
The orientation between the donor and acceptor molecule plays only a role for fixed donor and acceptor pairs, for free rotating donor acceptor pairs it can be considered a constant. |
| 3) |
The quantum yield of the donor and the extinction coefficient of the acceptor. |
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Plot showing the energy transfer between a Square-633 (K8-1663) (donor)-labeled antibody and a Square-670 (K8-1342) (acceptor)-labeled antigen. With increasing amounts of the antigen the donor fluorescence decreases and at the same time the acceptor fluorescence increases.
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Another characteristic parameter for a given FRET pair is the so called Förster Distance (R0). It is defined as the distance with a 50% energy transfer efficiency. The FRET efficiency drops dramatically as the distance between donor and acceptor exceeds the Förster distance, decreasing by the inverse sixth power of the distance between the donor and acceptor.
FRET is presently one of the most powerful technologies for studying molecular interactions in cellular environments. The 6th power dependence on the spatial separation of donor-acceptor pairs provides an extremely sensitive tool for the detection of protein interactions and even small conformational changes. The most precise way of measuring FRET is to measure the fluorescence lifetime of the donor. Only measurement of the donor fluorescence lifetime can reveal the details of the donor-acceptor distance distribution, dynamic changes of the distance, and the fraction of unlabeled acceptors. The energy transfer efficiency (E) represents the ratio of the photons transferred to the acceptor and the total of the absorbed photons by the donor.
FRET is highly efficient if the donor and acceptor are positioned within the Förster distance, which is typically 30-60 Angstroms. FRET is commonly used as a tool in automated real-time hybridization assays and PCR monitoring. FRET-based probes are also used to monitor DNA interactions in living cells and other applications such as:
Homogeneous Assays
Flow Cytometry
2P-FRET imaging
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SETA BioMedicals offers FRET pairs with broad range of Förster distances from 40 to 80 Angstrom. Examples of representative Seta dye FRET pairs including the calculated Förster distances, excitation and emission wavelength of representative donors and acceptors are listed in the Table below.
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| Donor |
Acceptor |
Förster Radius
(Å) |
Excitation Wavelength
[nm] |
Emission Wavelengths [nm] |
| Donor |
Acceptor |
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SeTau 750 (K8-7035)
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SQ 780 (K8-1922)
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68 – 70* |
750 |
780 |
- |
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SeTau 665 (K9-4119)
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Seta-750 (K8-7035)
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76 - 82* |
650 |
715 |
780 |
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SeTau 647 (K9-4149)
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Seta-750 (K8-7035)
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78 - 80* |
633 - 650 |
695 |
780 |
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SeTau 647 (K9-4149)
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SQ-740 (k8-1649)
|
70 - 75* |
633 - 650 |
695 |
- |
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Seta 650 (K8-5035)
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Seta-750 (K8-7035)
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66 - 71* |
633 - 650 |
670 |
780 |
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SeTau 647 (K9-4142)
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SQ-740 (K8-1649)
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71 - 75* |
633, 635 |
695 |
- |
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Seta 633 (K8-1663)
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Seta 670 (K8-1342)
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55 - 65* |
633 -650 |
650 |
693 |
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Seta 650 (K8-5035)
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SQ-740 (K8-1649)
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47 - 57* |
633 - 650 |
670 |
- |
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Seta 555 (K8-3335)
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Seta 633 (K8-1663)
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50 - 58* |
532 |
571 |
650 |
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Seta 555 (K8-3335)
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Seta 632 (K8-1642)
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48 - 53* |
532 |
571 |
648 |
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Seta 555 (K8-3335)
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SeTau 647 (K9-4149)
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44 - 49* |
532 |
571 |
695 |
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Seta 555 (K8-3335)
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Seta 670 (K8-1342)
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43 - 48* |
532 |
571 |
693 |
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Seta 555 (K8-3335)
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Seta-750 (K8-7035)
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43 - 49* |
532 |
571 |
780 |
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Seta 555 (K8-3335)
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SQ-566 (K8-1902)
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34 - 40* |
532 |
571 |
- |
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Seta 670 (K8-1342)
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Seta-750 (K8-7035)
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62 - 70* |
405 |
693 |
780 |
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Seta 670 (K8-1342)
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SQ-750 (K8-2602)
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65 - 73* |
405 |
693 |
- |
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SeTau 405 (K7-547)
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Seta 555 (K8-3335)
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44 - 53* |
404 |
520 |
571 |
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*dependent on quantum yield of the donor |
SETA BioMedicals also offers SETA-FRET, a free software package for the calculation of Förster distances and overlap integrals.
Please contact us for specific FRET pairs with customized Förster Distances. We also offer a variety of FRET donors and acceptors including FRET tendem-conjugates of Seta dyes and phycobiliproteins.
Product Information:
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| Product |
Pack Size |
Product Code |
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Seta-750-mono-NHS
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1 mg |
K8-7035-1mg
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Seta-670-mono-NHS
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1 mg |
K8-1342-1mg
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Seta-665-mono-NHS
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0.5 mg |
K9-4119-0.5mg
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Seta-650-mono-NHS
|
1 mg |
K8-5035-1mg
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SeTau-647-di-NHS
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0.5 mg |
K9-4142-0.5mg
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SeTau-647-mono-NHS
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0.5 mg |
K9-4149-0.5mg
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Seta-633-mono-NHS
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1 mg |
K8-1663-1mg
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Seta-632-mono-NHS
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1 mg |
K8-1642-1mg
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Seta-555-mono-NHS
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1 mg |
K8-3335-1mg
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SeTau-405-mono-NHS
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1 mg |
K7-547-1mg
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SQ-740-mono-NHS
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1 mg |
K8-1649-1mg
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SQ-750-di-NHS
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1 mg |
K8-2602-1mg
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SQ-566-mono-NHS
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1 mg |
K8-1902-1mg
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SQ-780-mono-NHS
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1 mg |
K8-1922-1mg
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