Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine‐reactive dye in combination with alkaline phosphatase‐ and horseradish peroxidase‐antibody conjugates

Abstract

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.

Identification of Lesional CD4+ CD25+ Foxp3+ Regulatory T Cells in Psoriasis

“…The advantages of the labeling technique used in the present study include the following: conjugations can be made on submicrogram quantities of a primary antibody, the primary antibody reactions are quantitative, fluorochrome labeling is completed in only minutes and is irreversible, multiple antibodies can be used in the same experiment, the fluorescence intensity of the cells can be adjusted by changing the ratio of labeling reagent to primary antibody, which permits using identical dyes to detect multiple targets in cells by flow cytometry, and, finally, a wide variety of fluorochromes is available [30, 33, 34]. …”

“…Whether T reg in psoriatic plaques are naturally occurring or adaptive cells, both of which express their suppressive function in different ways, remains to be elucidated. This is important, because natural and adaptive T reg might function in different immunological settings, depending for example on the context of antigen exposure and the nature of the inflammatory response [13, 34]. …”

Characterization of dynamic and steady‐state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry

“…Western blotting was performed as described [12], using a three buffer system. For the detection of NDUFS 4, 10 mg of Complex I was separated by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon P polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA).…”

Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

“…Factor VIII expressing cells were double stained for cell surface and intracellular rFVIII using the Zenon technology while BHK-21 native cells served as the negative control. The Zenon technique involves the conjugation of primary antibodies with fluorescent labeled Fab fragments of secondary antibodies directed against the primary antibody's Fc regions (Martin et al, 2003). With this technique it is possible to label a primary antibody quantitatively and rapidly with large fluorescent dyes such as R-phycoerythrin (R-PE) that cannot be readily labeled by covalent conjugation.…”