Cascade blue

The following sections discuss the most important Cascade Blue derivatives that are available for covalent modification purposes. 

Amine-Reactive Cascade Blue Acetyl Azide... 

One Cascade Blue derivative is available for creating linkages with amine-containing molecules. The acetyl azide functionality of this reagent reacts with primary amines at ambient temperatures or below to create amide bond derivatives (Lanier and Recktenwald, 1991 Oparka et al.,... 

Figure 9.37 The basic structure of Cascade Blue fluorophores.

Figure 9.38 The acetyl azide group of this Cascade Blue derivative has dual functions. It can react with amine groups to form amide bonds, or it can be converted to an isocyanate at high temperatures to couple with hydroxyl functional groups, creating a carbamate linkage.

At elevated temperatures (80°C in DMF), the acetyl azide group rearranges to form an isocyanate that can react with hydroxyl-containing molecules to form a urethane linkage (Figure 9.38). The Cascade Blue urethane derivatives of macromolecules are extremely fluorescent and can be detected down to femtogram quantities (Takadate et al., 1985). 

This fluorophore has excitation maxima at 375 and 400 nm and an emission maximum at 410 nm. The small Stoke s shift may create some difficulty in discrete excitation without contaminating the emission measurement with scattered or overlapping light. The extinction coefficient of the molecule in water is about 27,000M 1cm 1. Cascade Blue and Lucifer Yellow derivatives can be simultaneously excited by light of less than 400 nm, resulting in two-color detection at 410 and 530 nm. 

Cascade Blue acetyl azide is soluble in aqueous solution, but the reactive azide group will hydrolyze and should be used immediately in a conjugation reaction. A concentrated stock solution may be prepared in water, dissolved quickly, and an aliquot quickly added to a buffered reaction medium. For aqueous reactions, a pH range of 7-9 is optimal. Avoid amine-containing buffers. 

Carboxylate-Reactive Cascade Blue Cadaverine and Cascade Blue Ethylenediamine... 

Cascade Blue cadaverine and Cascade Blue ethylenediamine both contain a carboxamide-linked diamine spacer off the 8-methoxy group of the pyrene trisulfonic acid backbone. The cadaverine version contains a 5-carbon spacer, while the ethylenediamine compound has only a 2-carbon arm. Both can be coupled to carboxylic acid-containing molecules using a carbodiimide reaction (Chapter 3, Section 1). Since Cascade Blue derivatives are water-soluble, the carbodiimide EDC can be used to couple these fluorophores to proteins and other carboxylate-containing molecules in aqueous solutions at a pH range of 4.5-7.5. The reaction forms amide bond linkages (Figure 9.39). 

These fluorophores have excitation maxima at 377-378 nm and at 398-399nm and emission maxima at 422-423 nm. The extinction coefficients of the molecules in water are about 27,000 M 1cm 1. The Cascade Blue derivatives can be used along with Lucifer Yellow... 

Figure 9.39 The side-chain primary amine group of this Cascade Blue derivative can be coupled to carboxylate-containing molecules using a carbodiimide reaction.

Figure 9.40 Cascade Blue hydrazide can be used to modify aldehyde-containing molecules to form hydrazone bonds.

Aldehyde/Ketone-Reactive Cascade Blue Hydrazide... 

Whitaker, J.E., fdaugland, R.P., Moore, P.L., Hewitt, P.C., Reese, M., and Haugland, R.P. (1991) Cascade blue derivatives Water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. 

Figure 13.16, Donors and acceptors used for glucose sensing. Donors (a) Cascade Blue-ConA (b) Texas Red-Con A (c) AMCA-ConA. Acceptors Malachite Green-dextran.

A UV laser is needed for exciting the blue-fluorescing agents, 4, 6-diamidino-2-phenyhndole (DAPI) and Hoechst 33342, which are DNA-intercalating stains, and for indo-1, a fluorescent calcium chelator dye. Violet diode lasers that are offered in some newer instruments accommodate fluorochromes such as Cascade Blue, Pacific Blue, and cyan fluorescent protein, and are also capable of exciting DAPI (Shapiro and Perlmutter 2001 Telford et al., 2003). 

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