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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:

 
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.

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
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.
Donor Acceptor Förster Radius
(Å)		 Excitation Wavelength
[nm]		 Emission Wavelengths [nm]
Donor Acceptor
68 – 70* 750 780 -
76 - 82* 650 715 780
78 - 80* 633 - 650 695 780
70 - 75* 633 - 650 695 -
66 - 71* 633 - 650 670 780
71 - 75* 633, 635 695 -
55 - 65* 633 -650 650 693
47 - 57* 633 - 650 670 -
50 - 58* 532 571 650
48 - 53* 532 571 648
44 - 49* 532 571 695
43 - 48* 532 571 693
43 - 49* 532 571 780
34 - 40* 532 571 -
62 - 70* 405 693 780
65 - 73* 405 693 -
44 - 53* 404 520 571
  *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:
Product Pack Size Product Code
1 mg
K8-7035-1mg
1 mg
K8-1342-1mg
0.5 mg
K9-4119-0.5mg
1 mg
K8-5035-1mg
0.5 mg
K9-4142-0.5mg
0.5 mg
K9-4149-0.5mg
1 mg
K8-1663-1mg
1 mg
K8-1642-1mg
1 mg
K8-3555-1mg
1 mg
K7-547-1mg
1 mg
K8-1649-1mg
1 mg
K8-2602-1mg
1 mg
K8-1902-1mg
1 mg
K8-1922-1mg

Pricing and Availability
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Product Specifications :
Excitation Light Sources Free Dye Dye Conjugate
Product Number Product Name 380405 436488532594635670SolventλAb max. [nm]ε [M–1
cm–1]		 λEm max. [nm]QY [%] FLT [ns] Carrier
PB 7.4		λAb max. [nm]λEm max. [nm]QY [%]FLT [ns]
                           
K7-545 SeTau-425-NHS • 425 4200 545 39 26.2 0.0 427 528 16
K7-547 SeTau 405 NHS • • PB 7.4 405 13800 518 80 9.3 BSA, D/P=8 406 518 20 7.6
K7-549 SeTau-404-NHS • • PB 7.4 402 10600 515 52 9.3 BSA, D/P=2 402 503 8.8
K8-1342 Seta-670-NHS • • • PB 7.4 667 179000 688 7 BSA, D/P=1 681 695 35 2.4
K8-1352 Square-660-NHS • • PB 7.4 658 182000 677 3 0.26 BSA, D/P=1 676 695 13 3.32
K8-1500 Square-655 • • • MeOH 628 177000 654 0.09 1 g/L BSA 652 672 80
K8-1626 Seta-633-di-NHS • PB 7.4 635 192000 644 12 0.7 IgG, D/P=1 639 650 33 3
K8-1642 Seta-632-NHS • PB 7.4 632 280000 641 6 IgG, D/P=1 637 647 18
K8-1663 Seta-633-NHS • PB 7.4 633 250000 644 7 0.3 IgG, D/P=1 637 647 26 1.8
K8-1669 Seta-635-NH-NHS • PB 7.4 638 136000 655 31
K8-1682 Seta-660-di-NHS • PB 7.4 661 151000 672 11 IgG, D/P=1 665 675 49
K8-3335 Seta-555-NHS • PB 7.4 556 155000 570 12 IgG, D/P=1 654 571 23
K9-4112 SeTau-655-di-NHS • PB 7.4 665 200000 673 25 0.9 IgG, D/P=1 657 675 31 1.8
K9-4113 SeTau-665-di-NHS • • PB 7.4 665 140000 715 62 3.1 IgG, D/P=1 664 709 43 3.3
K9-4142 SeTau-647-di-NHS • PB 7.4 650 368000 694 65 3.2 IgG, D/P=0.5 649 691 58 3.2
K1-204 SeTau-380-NHS • Water 270 23800 480 56 32.5 BSA, 383 468 44 30.6
K8-5035 Seta-650-NHS • PB 7.4 651 172000 671 28 IgG, D/P=1 654 673 37
K8-7035 Seta-750-NHS • PB 7.4 750 235000 779 22 IgG, D/P=1 755 783 24
K8-1344 Seta-665-NHS • • • PB 7.4 667 118000 683 3 BSA, D/P=1 682 697 38 3
K8-1367 Seta-670-NH-di-NHS • • PB 7.4 672 93000 696 15 2 IgG, D/P=1 684 703 7
K8-1377 Seta-690-NH-di-NHS • • PB 7.4 693 113000 714 14 BSA, D/P=1 711 726 14 1.5
K8-1762 Seta-635-B-di-NHS • • • • PB 7.4 634 131000 656 8 0.5 BSA, D/P=0.5 650 672 41 3
K8-1782 Seta-660-B-di-NHS • • • • PB 7.4 659 132000 679 9 BSA, D/P=1 672 695 28 2.2
K8-1667 Seta-635-NH-di-NHS • PB 7.4 640 188000 656 33 1.8 IgG, D/P=1 646 663 21
K8-5036 Seta-650-maleimide • PB 7.4 652 210000 672 28
K8-7038 Seta-750-Maleimide • PB 7.4 752 235000 779 22 IgG, D/P=1 755 783 24
K9-4149 Seta-647-NHS • PB 7.4 649 200000 695 61 3.2 IgG, D/P=1 648 694 59 3.3
K9-4119 SeTau-665-NHS • • • PB 7.4 664 160000 712 53 3.1 IgG, D/P=1.2 653 716 50 3.3
K8-1384 Square-690-mono-NHS • • PB7.4 687 130000 703 13 0 IgG, D/P = 1 704 716 72
 
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