Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes

A two-color fluorescence detection method is described based upon covalentl y coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of tar get proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethyla cridin-2-one-7-yl(DDAO)phosphate in combination with alkaline-phosphatase-c onjugated reporter molecules. This results in all proteins in the profile b eing visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detect ion system is broadly compatible with a wide range of analytical imaging de vices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-are sources equipped with approp riate excitation/emission filters, and dual laser gel scanners outfitted wi th a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection meth od permits detection of low nanogram amounts of protein and allows for unam biguous identification of target proteins relative to the entire protein pr ofile on a single electroblot, obviating the need to run replicate gels tha t would otherwise require visualization of total proteins by silver stainin g and subsequent alignment with chemiluminescent or colorimetric signals ge nerated on electroblots.