Fluorescent protein tracers: the unreacted fluorescent material in fluorescein conjugates and studies of conjugates with other green fluorochromes.

Protein conjugates with fluorescein isocyanate and isothiocyanate contain unreacted fluorescent material (U.F.M.) which contributes to specific immunological staining but is troublesome because it also stains non-specifically. Attempts have been made to find an alternative to fluorescein isocyanate and isothiocyanate conjugates, with similar green fluorescence but free from U.F.M. and more easily prepared. Conjugates were made with fluorescein and certain derivatives of fluorescein in the form of the acid chlorides, `reactive' dyes, a dichlorocyclopropyl derivative of a stilbene sulphonic acid (R 8065/1, Geigy), and 3-hydroxypyrene 5-8-10 trisulphonic acid; none proved satisfactory. At the present time, green fluorescent protein tracers are probably best prepared from fluorescein isothiocyanate. The properties and nature of the U.F.M. in fluorescein conjugates have been investigated; it accounts for nearly half of their fluorescence after full dialysis and two extractions with tissue powder. Extraction with powdered activated charcoal removes the U.F.M. almost completely but the staining intensity of the conjugates is so reduced that the procedure cannot be recommended for routine use. This property of charcoal provides a simple means of testing fluorescein conjugations: a sample from a satisfactory conjugate should retain substantial fluorescence after such extraction.