| Assay Method Information | |
| | Tpl2/Cot ELISA Assay |
| Description: | Tpl2/Cot activity was directly assayed using GST-MEK1 as a substrate. The phosphorylation on serine residues 217 and 221 of GST-MEK1 was detected by an ELISA. The kinase reaction was carried out in 96-well plates. After the reaction was stopped, the entire reaction mix was transferred to the detection plate, a 96-well Immunosorb plate that had been precoated with anti-GST antibody (Amersham), and then subsequently incubated with anti- phospho-MEK1 antibody (Cell Signaling), and DELFIA Europium (Eu) labeled goat anti-rabbit IgG (Perkin-Elmer). The Eu signal was measured in a Wallac Victor Multilabel Counter. Data were imported into Excel and IC50 calculations were performed using the Xlfit (IDBS) software package. |
| Affinity data for this assay | |
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