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ab126572 involves incorporation of BrdU into cells cultured in microtiter plates using the cell layer as the solid phase. During the final 2 to 24 hours of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU cells must be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse antibody is added, which binds to the Detector Antibody.
The horseradish peroxidase catalyzes the oxidation of diacylhydrazides. The reaction product, in its excited state, decays yielding light in the process. This reaction has a large dynamic range spanning many logs of relative luminescence. The intensity of the luminescence is proportional to the amount of incorporated BrdU in the cells. The reaction is quantified using a luminometer and is plotted as relative light units per second (RLU/s).
The resultant assay is sensitive, rapid, easy to perform and applicable to high sample throughput. In addition to evaluation of cell proliferation, information such as cell number, morphology and analysis of cellular antigens can be obtained from a single culture.
|BrdU Reagent||1 x 15µl|
|Conjugate Diluent||1 x 25ml|
|Fixing Solution||2 x 20ml|
|Peroxidase Goat anti-mouse IgG (2000X)||1 x 15µl|
|Plate wash concentrate (50X)||1 x 90ml|
|Prediluted anti-BrdU detecting antibody||1 x 20ml|
|Reaction Buffer||1 x 24ml|
|Substrate||1 x 800µl|
Our Abpromise guarantee covers the use of ab126572 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Indirect ELISA||Use at an assay dependent concentration.|
ab126572 has not yet been referenced specifically in any publications.