Principle of Assay
This B-RAF (phospho Ser446) Cell Based ELISA Kit allows for the detection of B-RAF (phospho Ser446) and the effects that certain stimulation conditions have on B-RAF (phospho Ser446) expression in different cell lines. Qualitative determination of B-RAF (phospho Ser446) concentration is achieved by an indirect ELISA format. In essence, the B-RAF (phospho Ser446) is captured by Anti-B-RAF (phospho Ser446) Antibodies which in turn are detected by HRP-conjugated secondary antibodies. Through this binding, the HRP enzyme (conjugated to the secondary antibody) can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of this B-RAF (phospho Ser446) Cell Based ELISA Kit, multiple normalization methods are described: 1) Anti-GAPDH Antibody is included to serve as an internal positive control in normalizing the target absorbance values. 2) Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method is used to determine cell density. After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) Anti-B-RAF Antibody is provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target.
