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Square Labels

Square dyes are in general more hydrophobic than Seta or SeTau dyes as they are more environment-sensitive. They typically exhibit lower quantum yields in aqueous environment and high quantum yields after binding to proteins or other biomolecules. Importantly the quantum yields and fluorescent lifetimes increase also upon covalent labeling to oligonucleotides, which makes them preferred labels also for oligos. In general Square Dyes are most suitable for fluorescence lifetime based applications wherein a small molecular weight analyte labeled with a Square dye interacts with larger biomolecules (proteins or lipids).

Excitation Light Sources Characteristics 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]
Medium λ abs
[nm]
ε
[M–1
cm–1]
λ em
[nm]
QY
[%]
FLT
[ns]
Buy
                               
K8-1352
K8-1352
Square-660-NHS
Square-660-NHS
NH2 PB 7.4 657 182000 676 3 0.27 BSA D/P=1.2 676 182000 695 13 3.32
K8-1357
K8-1357
Square-680-NHS
Square-680-NHS
NH2 MeOH 668 208000 690 10 CHCl3 683 202000 701 47
K8-1407
K8-1407
Square-650-pH-NHS
Square-650-pH-NHS
NH2 PB 5.6 653 135000 671 16 1.17 IgG, D/P=0.8
pH=2.0
662 135000 677 7 1.52
K8-1612
K8-1612
Square-635-NHS
Square-635-NHS
NH2 pH 7.4 634 180000 646 9 BSA, D/P=1.0 649 663 29
K8-1613
K8-1613
Square-635-Maleimide
Square-635-Maleimide
SH pH 7.4 636 180000 649 8

The table below provides data of the expected changes in lifetime for Square-660-NHS. It is important to note that the observed lifetime change of a fluorescently labeled ligand will strongly depend on the nature and size of the biomolecule it binds to:

 

Dye Dye – BSA Conjugate
D/P = 1.2
Δτ [ns]
τ(Dye conj.) – τ(Dye)
Product Number
(Specs Sheet)
Product Name
(Product Info)
τmean
at 25 °C [ns]
τmean
at 25 °C [ns]
K8-1352 Square-660-NHS 0.27 3.32 3.05

 

SETA BioMedicals offers several of these environment-sensitive FLT labels: all are excitable with a 633, 635 or 647 nm laser source (for more information see lifetime applications).

 

Comparison of the frequency responses of   Square-660 before and after binding to BSA
Comparison of the frequency responses of
  Square-660 before and after binding to BSA.
(τfree = 290 ps; τBSA bound = 3.32 ns)
Comparison of the intensity decays of   Square-660 before and after binding to BSA
Comparison of the intensity decays of
  Square-660 before and after binding to BSA.
(τfree = 290 ps; τBSA bound = 3.32 ns)

Lifetime-based Hybridization Assay with Square-660-labeled oligonucleotide:

Importantly Square dyes exhibit a large increase in quantum yields and lifetime upon binding to oligonucleotides. Square 660 was also successfully labeled to a single-stranded oligonucleotide and the hybridization with the complementary nucleotide lead to a change in its fluorescence lifetime from 3 ns to 1.8 ns that could be easily measured with lifetime-based micro-plate reader from TECAN (see application note below).

 

Intensity decays of Square-660-oligo before and after binding to complementary oligonucleotide<
Intensity decays of Square-660-oligo before
and after binding to complementary oligonucleotide
(τsingle = 3 ns; τdouble = 1.8 ns)
Lifetime-based detection of SNPs. Detection is based on the change of the fluorescence lifetime of the label Square-660
Lifetime-based detection of SNPs. Detection is
based on the change of the fluorescence lifetime
of the label Square-660

pH-measurement in cells: Probes and labels such as Square-650-pH exhibit pKa’s in the physiologically pH range and are very useful to determine the intercellular pH in cells. For more information about these dyes we refer you to the specs sheet of Square-650-pH and the published literature [9].

 

Fluorescence spectrum of Square-650-pH vs. pH
Fluorescence spectrum of Square-650-pH vs. pH
Titration curve of Square-650-pH
Titration curve of Square-650-pH

 

Measurement of endocytosis and internalization of surface labeled proteins with Square-650-pH. Importantly this is the only probe that enables measurement of endocytic events based on the measurement of fluorescence lifetime (FLIM) which is known to be more robust as compared to other parameters. Please make sure that you read the instructions on the specifications sheet to insure proper handling of the compound and labeled biomolecules.

 

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Fluorescence intensity of Square-650-pH free in solution and conjugated to IgG and E.Coli cells
Application of Square-650-pH to investigate phagocytosis events
Application of Square-650-pH to investigate phagocytosis events

 

Product Number
(Specs Sheet)
Product Name
(Product Info)
K8-1407 Square-650-pH-NHS

Square-680-NHS is suitable for labeling in hydrophobic environments.

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