Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

Abstract

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures

“…the relative expression levels of proteins which overlay in dot blots or may exhibit similar or identical molecular masses in Western blot analysis. Several methods have been described for simultaneous or serial determination of different antigens on one immunoblot which allow protein discrimination by using various chromogenic, fluorescence and chemiluminescence reactions (Hsu and Soban, 1982;Lee et al, 1988;Poor et al, 1988;Schilling and Aletsee-Ufrecht, 1989;Kondoh et al, 1998;Martin et al, 2003;Kuczius et al, 2010). However, the techniques are limited when antigenic determinants overlapping in molecular masses.…”