NHS-fluorescein

The fluorescent properties of NHS-fluorescein are similar to FITC. The wavelength of maximal absorbance or excitation for the reagent is 491nm and its emission maximum is 518nm, exhibiting a visual color of green (Sheehan and Hrapchak, 1980). Its molar extinction coefficient at 491 nm in a pH 8.0 buffer environment is Other components in... 

NHS-fluorescein 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 if protected from light. Reaction conditions should be maintained at the optimal reactivity for NHS esters—pH 7-9. 

Figure 9.6 NHS-fluorescein reacts with amine-containing compounds via its NHS ester to form amide bonds.

The following procedure is a suggested method for using NHS-fluorescein to label immunoglobulins. 

Dissolve 0.5mg of NHS-fluorescein (Thermo Fisher) in 0.5 ml DMSO. Protect from light. 

In a darkened lab, slowly add 50-100pi of the NHS-fluorescein solution to the antibody solution, while mixing. Protect from light by wrapping the reaction vessel in aluminum foil. 

Remove unreacted NHS-fluorescein and reaction by-products by gel filtration or dialysis. Continue to protect all labeled protein solutions from light. 

The optimal reaction conditions for derivatizing the tagging RNA with a biophysical label depend on the NHS ester used (Fig. 5.6A). Derivatizations with NHS-biotin or NHS-TAMRA can be performed under the same conditions derivatization with NHS-fluorescein was optimal with different conditions. 

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