SETA BioMedicals

Square Dyes

Square Dyes

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).

The table below provides data of the expected changes in lifetime for Square 660 -NHS (K8-1352). 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 - Conjugate	Δτ [ns] τ(Dye conj.) –τ(Dye)
Product Name	Product Code	τ_mean [ns]	D/P ratio	τ_mean [ns]	Δτ [ns] τ(Dye conj.) –τ(Dye)
Square 660 NHS	K8-1352	0.29	1.2	3.3	3.01

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 protein (τ_free = 250 ps; τ_bound = 2.3 ns)	Comparison of the intensity decays of Square 660 before and after binding to protein

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

Importantly square dyes exhibit a large increase in quantum yeilds 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 (τ_single= 3 ns; τ_double= 1.8 ns)	Lifetime-based detection of SNPs.Detection is based on the change of the fluorescence lifetime of the label Square 660.

http://www.tecan.com/platform/content/element/1414/Tecan_ULTRAEvolution_AppNote_DNADetection.pdf

pH-measurement in cells: Probes such as K8-1405 (carboxyl version of K8-1407) and K8-1407 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 K8-1407 and the published literature.

Measurement of endocytosis and internalization of surface labeled proteins with K8-1407. 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.

Product	Pack Size	Product Code
Square 650-pH-NHS	1 mg	K8-1407-1mg
Square 650-pH-COOH	1 mg	K8-1405-1mg

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Dinitro-reductase enzyme activity in cells: Our novel dye-substrates containing dinitro-benzyl-substituents show favorable properties for use of imaging of dinitro-reductase enzyme activity in cells.

Visualization of nitro-reductase (DNT) enzyme activity in cells can be used as a non-invasive, live-cell reporter system. A cell permeable fluorogenic substrate is used to report on the enzyme activity, which is based on the increase of the fluorescence signals upon reduction of the nitro-group on the enzyme substrate (Figure). This non-destructive method offers the ability to screen out non-responding cells, analysis of individual cell responses, and measurement of a second fluorescent event occurring within the same cell.

Product	Pack Size	Product Code
DNB-Square 670	1 mg	K8-1359-1mg

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Square-650-mono-NHS (K8-1261) and Square-670-mono-NHS (K8-1357) are suitable for labeling in hydrophobic environments.

Product Specifications:


			Excitation Light Sources								Free Dyes					Dye Conjugate
Product Number	Product Name	Target Group	380	405	436	488	532	594	635	670	Solvent	λ_Ab max. [nm]	ε [M^–1cm^–1]	λ_Em max. [nm]	QY [%]	Carrier PB 7.4	λ_Ab max. [nm]	λ_Em max. [nm]	QY [%]
K8-1252	Square-635-di-NHS	NH₂	•						•		PB 7.4	636	216000	648	6	BSA, D/P=0.5	652	665	20
K8-1352	Square-660-NHS	NH₂		•						•	PB 7.4	658	182000	677	3	BSA, D/P=1	676	695	13
K8-1357	Square-685-NHS	NH₂	•	•					•	•	CHCl3	683	208000	703	45
K8-1261	Square-650-NHS	NH₂	•	•					•		CHCl₃	649	138000	664	17
K8-1320	Square-670-di-NHS	NH₂		•						•	PB 7.4	667	188000	685	7	BSA, D/P=1	682	701	23
K8-1752	Square-635-B-di-NHS	NH₂		•	•				•		PB 7.4	628	103000	649	5	BSA, D/P=1	645	668	27
K8-1407	Square-650-pH-NHS								•		PB 7.4	653	135000	671	16	IgG, D/P=1	662	677	7
K8-1384	Square-690-mono-NHS	NH2		•						•	PB7.4	687	130000	703	13	IgG, D/P = 1	704	716	72