Square Labels

Square dyes are in general more hydrophobic than Seta or SeTau dyes as they are more environment-sensitive. They exhibit lower quantum yields in aqueous environments and higher quantum yields in non aqueous environments or after binding to proteins and other biomolecules. Importantly, the quantum yields and fluorescent lifetimes of Square dyes also increase upon covalent attachment to oligonucleotides, which makes them preferred labels also for oligos. They are most suitable for fluorescence lifetime based applications where a labeled, low-molecular-weight analyte interacts with a larger biomolecule (protein, oligo or lipid).

			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	182,000	676	3	0.27	BSA D/P=1.2	676	182,000	695	13	3.32
K8-1357 K8-1357	Square-680-NHS Square-680-NHS	NH2	•	•					•	•					MeOH	668	208,000	690	10		CHCl3	683	202,000	701	47
K8-1407 K8-1407	Square-650-pH-NHS Square-650-pH-NHS	NH2						•	•	•					PB 5.6	653	135,000	671	16	1.17	IgG, D/P=0.8 pH=2.0	662	135,000	677	7	1.52
K8-1612 K8-1612	Square-635-NHS Square-635-NHS	NH2						•	•						pH 7.4	634	180,000	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	180,000	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).

 

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 the lifetime-based micro-plate reader from TECAN (see below).

 

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].

 

 

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 ensure proper handling of the compound and labeled biomolecules.

 

 

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

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