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Single Molecule Applications

Excitation Light Sources Characteristics
Product Number
(Specs Sheet)
Product Name
(Product Info)
Target Group 380 405 436 488 532 594 635 650 680 700 750 780 Medium λ abs
[nm]
ε
[M –1
cm–1]
λ em
[nm]
QY
[%]
FLT
[ns]
Buy
                   
K7-567
K7-567
SeTau-405-Azide
SeTau-405-Azide
triple-CC PB 7.4 405 13800 518 80 9.3
K8-1342
K8-1342
Seta-670-NHS
Seta-670-NHS
NH2 PB 7.4 667 180000 688 7 0.42
K7-545
K7-545
SeTau-425-NHS
SeTau-425-NHS
NH2 PB 7.4 425 4200 545 39 26.2
K8-1641
K8-1641
Seta-632-Maleimide
Seta-632-Maleimide
SH PB 7.4 633 270000 642 5
K8-1642
K8-1642
Seta-632-NHS
Seta-632-NHS
NH2 PB 7.4 632 280000 641 6
K8-1663
K8-1663
Seta-633-NHS
Seta-633-NHS
NH2 PB 7.4 633 250000 644 7 0.25
K8-1672
K8-1672
Seta-646-NHS
Seta-646-NHS
NH2 PB 7.4 646 207000 656 10 0.38
K8-1696
K8-1696
Seta-633-Azide
Seta-633-Azide
triple-CC PB 7.4 633 250000 644 7
K8-3335
K8-3335
Seta-555-NHS
Seta-555-NHS
NH2 PB 7.4 555 155000 570 7
K8-3345
K8-3345
Seta-555-DBCO
Seta-555-DBCO
N3 PB 7.4 555 155000 570 7
K8-3346
K8-3346
Seta-555-Azide
Seta-555-Azide
triple-CC PB 7.4 555 155000 570 7
K8-5035
K8-5035
Seta-650-NHS
Seta-650-NHS
NH2 PB 7.4 651 200000 671 28
K8-5036
K8-5036
Seta-650-Maleimide
Seta-650-Maleimide
SH PB 7.4 652 200000 672 28
K8-5045
K8-5045
Seta-650-DBCO
Seta-650-DBCO
N3 PB 7.4 653 200000 674 28
K8-5046
K8-5046
Seta-650-Azide
Seta-650-Azide
triple-CC PB 7.4 651 200000 671 28
K9-4119
K9-4119 new
SeTau-665-NHS
SeTau-665-NHS
NH2 PB 7.4 664 160000 712 53 3.1
K9-4142
K9-4142
SeTau-647-di-NHS
SeTau-647-di-NHS
NH2 PB 7.4 650 200000 694 65 3.2
K9-4145
K9-4145
SeTau-633-Ethyl-Ester
SeTau-633-Ethyl-Ester
CHCl3 634 88000 683 68
K9-4148
K9-4148
SeTau-647-Maleimide
SeTau-647-Maleimide
SH PB 7.4 648 200000 692 45 3.2
K9-4149
K9-4149
SeTau-647-NHS
SeTau-647-NHS
NH2 PB 7.4 649 200000 695 61 3.2
K9-4150
K9-4150
SeTau-647
SeTau-647
PB 7.4 647 211000 693 59 3.1
K8-1341
K8-1341
Seta-670-Maleimide
Seta-670-Maleimide
SH PB 7.4 667 180000 688 7
K7-548
K7-548
SeTau-405-Maleimide
SeTau-405-Maleimide
SH PB 7.4 405 13800 518 51 9.1
K8-7522
K8-7522
Seta-750-NHS
Seta-750-NHS
NH2 PB 7.4 753 230000 780 14

 

Some of our Seta dyes e.g. Seta-670-NHS show extremely low blinking effects at the single molecule level and high photostability. These labels exhibit easy conjugation chemistry and are available in both NHS and maleimide forms. Below is an example of a single molecule application that was done with Seta-670-NHS and a commercially available antibody.

Our cyanine-based dyes are more photostable compared to the conventional Cy dyes and Seta-555, Seta-650 and Seta-750 exhibits even higher photostability compared to Alexa dyes and are therefore well suited for single molecule measurements.

Another class of dyes which promises excellent performance for single-molecule applications are Squaraine-rotaxanes (SeTau dyes). These combine high photostability and high sensitivity. They are available in hydrophobic and hydrophilic forms.

 k8

Relative photostability of Seta-555 and Seta-650 compared to Cy3, Cy5 and Alexa 647 upon irradiation with a 500 W halogen lamp

 k8

Characteristic single-molecule time trajectories for Seta-670-labeled IgG coated on glass with a dye-to-protein ratio of 3. Insets to trajectories show values of fluorescence decay times for the specific label [17]

Single Molecule FRET

Fluorescence resonance energy transfer (FRET) is used to obtain distance information from 10 to 100 Ǻ, a range suitable for studying the global structure and interactions of biomolecules. Nevertheless, some conformational changes are difficult to detect using ensemble FRET. The development of single-molecule spectroscopy makes it possible to probe the conformational dynamics and interactions of biological systems at single-molecule level. To date the most popular fluorescent reporter molecules for single molecule studies are still small size organic molecules. Besides small size and brightness an ideal fluorescent reporter for single-molecule studies should exhibit good photostability and a low tendency for aggregation including easy conjugation chemistry. FRET pairs should also have an adequate shift between the donor and acceptor emissions and similar brightness [18].

Importantly Seta-670-NHS has a significant overlap integral between its absorption and emission spectra. Its Förster distance (R0) for homo-FRET was calculated to be about 50 A, a distance comparable to the size of the antibody. The homo-FRET is expected to occur already when an antibody is labeled with only two fluorophores and increases quickly with the number of labels. Analysis of single molecule traces shows an interesting behavior where efficient non-radiative excitation energy transfer and self-quenching already manifests itself with only 2 or 3 labels. Detailed analyses of the overall and average residence times reveal that multiple labeling with fluorophores, such as Seta-670-NHS, could be a good approach for increasing the number of available photons and extending the overall observation time to study/observe binding at the single antibody level. Intrinsically, the signal and lifetime of the individual, intermediate fluorescently labeled species strongly depend on the number of labels, but the average residence time for each single species is similar. Apparently due to energy transfer the change in fluorescence intensity is compensated by the change in average fluorescence lifetime. Contrary to ensemble measurements where over-labeling is commonly recognized as a problem, this approach appears to have significant advantages for single antibody (protein) studies.

For additional FRET pairs we refer you to FRET applications, where we provide a more comprehensive list of donors and acceptors including the calculated Förster distances.

 

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