Introduction

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates; however, fluorescein and rhodamine (Table 1) are the most commonly used.

The isothiocyanate derivatives of fluorescein and rhodamine are widely used to label antibodies. They react with the amino groups of the immunoglobin G (IgG) molecule under alkaline conditions and a molar excess of about 20 is usually optimal. More recently, N-

Table 1

Properties of Fluorescein and Rhodamine

Properties of Fluorescein and Rhodamine

Table 1

Molecular

Excitation

Emission

Fluorochrome

weight*

(nm)

(nm)

Fluorescein

389

495

520

Tetramethyl

443

555

575

rhodamine

*The molecular weight given is that of the isothiocyanate derivative

*The molecular weight given is that of the isothiocyanate derivative hydroxysuccinimide derivatives have become available for the same purpose and they use milder conditions. In both cases, the reaction is quenched by the addition of ethanolamine and excess labeling reagent is removed by gel filtration. The extent of conjugation may be determined by absorbance measurements.

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