Principle of Assay
This Smad1 + 5 + 9 Cell Based ELISA Kit allows for the detection of Smad1 + 5 + 9 and the effects that certain stimulation conditions have on Smad1 + 5 + 9 expression in different cell lines. Qualitative determination of Smad1 + 5 + 9 concentration is achieved by an indirect ELISA format. In essence, the Smad1 + 5 + 9 is captured by Anti-Smad1 + 5 + 9 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 Smad1 + 5 + 9 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.
