Principle of Assay
This CrkII (phospho Tyr221) Cell Based ELISA Kit allows for the detection of CrkII (phospho Tyr221) and the effects that certain stimulation conditions have on CrkII (phospho Tyr221) expression in different cell lines. Qualitative determination of CrkII (phospho Tyr221) concentration is achieved by an indirect ELISA format. In essence, CrkII (phospho Tyr221) is captured by Anti-CrkII (phospho Tyr221) Antibodies while Dye 1-conjugated and Dye 2-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the dye conjugated to the secondary antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values. Anti-CrkII Antibody is also included for normalization purposes. The RFU values obtained for the non-phosphorylated target can be used to normalize the RFU value for the phosphorylated target.
