![]() ![]() The prestained molecular weight marker proteins, however, are not detected by the chemiluminescent reaction and are therefore not displayed on the X-ray film, making it necessary to manually chart the protein marker bands on the film (or to overlay the CCD camera captured picture of the emitted light with the one of the stained marker captured under daylight) in order to estimate the molecular weight of the detected protein bands. The major advantage of chemiluminescence over fluorescence detection is the signal amplification due to the enzyme catalyzed reaction, allowing the detection of minute amounts of the target protein. The emitted light is detected either on X-ray films or with the help of CCD-based camera systems. The most widely used enzyme for Western blot detection is horseradish peroxidase (HRP), which catalyzes the chemiluminescent oxidation of luminol. These proteins of interest, however, have to be visualized by specific antibodies that are coupled to fluorophores or enzymes catalyzing a chemiluminescent reaction. Almost all of the commercially available molecular weight markers consist of proteins prestained with vinyl sulfone dyes, also known under their trademark name as Remazol ® dyes, which provide visible reference points for the proteins of interest 3, 4, 5. To estimate the relative molecular weight of a specific protein, protein molecular weight markers are separated side-by-side with the protein sample. The most widely used method for the analysis of proteins is sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) 1, which is often followed by transferring the proteins to a membrane, where the proteins get immobilized and detected with antibodies, generally referred to as Western blot analysis 2. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. ![]() To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. ![]() Prestained proteins are used as molecular weight standards in protein electrophoresis. Western blotting is one of the most widely used techniques in molecular biology and biochemistry. ![]()
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