NHS-rhodamine

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

NHS-rhodamine is an amine-reactive fluorescent probe that contains a carboxy-succinimidyl ester group off the No. 5 or 6 carbons on rhodamine s lower-ring structure (Kellogg et al., 1988). The 5- and 6-isomers are virtually identical in their reactivity and fluorescent characteristics. Similar to TRITC (described previously), NHS-rhodamine can be used to label proteins and other macromolecules that contain primary amine groups. The isomeric forms of the fluorescent probe are available in mixed and purified forms (Invitrogen, Thermo Fisher). The pure forms are... 

Figure 9.15 NHS-rhodamine can be used to label amine-containing molecules via its NHS ester group.

The fluorescent properties of NHS-rhodamine are similar to TRITC. The wavelength of maximal absorbance or excitation for the reagent is 544 nm and its emission maximum is 576 nm, exhibiting a visual color of orange-red. Its molar extinction coefficient at 546 nm in a methanol environment is 63,000M 1cm 1. Other components in solution as well as the pH (in aqueous buffers) can change this value. 

NHS-rhodamine is insoluble directly in aqueous solution and should be dissolved in organic solvent prior to addition of a small aliquot to a buffered reaction medium. Concentrated stock solutions may be prepared in DMSO or DMF. Such solutions are relatively stable for short... 

The following generalized protocol relates to the labeling of IgG with NHS-rhodamine. Optimization of the level of rhodamine incorporation may have to be done with other proteins or other macromolecules. 

Dissolve NHS-rhodamine at a concentration of 1 mg/ml in DMSO. Protect from light. 

In a darkened lab, slowly add 50-100pi of the NHS-rhodamine solution to each ml of the antibody solution with mixing. Wrap the vessel with aluminum foil to protect from light. 

Remove unreacted NHS-rhodamine and reaction by-products by gel filtration or dialysis. Lissamine Rhodamine B Sulfonyl Chloride... 

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