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ab126556 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 conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is quantified using a spectrophotometer.
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|
|Stop Solution||1 x 25ml|
|Substrate||1 x 25ml|
Our Abpromise guarantee covers the use of ab126556 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.|
Cell proliferation measured in fibroblasts showing cell amounts vs. optical densities