Principle of Assay
This CD5 (phospho Tyr453) Cell Based ELISA Kit allows for the detection of CD5 (phospho Tyr453) and the effects that certain stimulation conditions have on CD5 (phospho Tyr453) expression in different cell lines. Qualitative determination of CD5 (phospho Tyr453) concentration is achieved by an indirect ELISA format. In essence, the CD5 (phospho Tyr453) is captured by Anti-CD5 (phospho Tyr453) 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 CD5 (phospho Tyr453) 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-CD5 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.
