Moreover due to their intrinsic binding affinities for the Ig Fc domain also primary antibodies directed against the desired target are bound by these markers reducing their availability for antigen detection. MagicMark TM XP Western Protein Standard, SuperSignal Molecular Weight Protein Ladder, both Life Technologies), which allow their detection with standard secondary antibodies however, these are species-specific IgG binding sites and therefore different protein marker ladders have to be matched to the appropriate secondary antibody used. Other marker proteins were engineered to contain immunoglobulin G (IgG) binding sites (e.g. LI-COR Odyssey, or GE Healthcare Typhoon). Protein molecular weight markers coupled to fluorescent dyes can be directly detected by Western blot analysis, but require expensive scanner equipment (e.g. The so-called Optiblot Luminol Pen (Abcam) is easy to use, but still requires the manual labeling of protein marker bands. This problem has been addressed several times, but all of the currently available systems have major disadvantages and restrictions that limit their usage. This process not only seems anachronistic in an otherwise high-tech research field but is intrinsically prone to human error, as the film is fitted to the membrane and has to be perfectly positioned to accurately copy the marker bands: first, reference points are often lacking since the contours of the membrane are not visible on the film, and second, any inaccuracy of the experimenter in mapping the shapes of the marker bands may directly affect data interpretation. 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.