Section8:Appendix 2. Linearity and Sensitivity of Cell-Based Western Assays

From Assay Guidance Wiki

HEK293 cells were transfected with a specific enayme substrate using standard procedures. The next day, transfected and control (not transfected) cells were harvested and mixed together in the proportions indicated. The mixed populations were seeded into individual wells of a 96-well plate and cultured for an additional 24h. Cells were serum starved for 2 h in medium containing 0.1% BSA before adding a specific enzyme inhibitor. After additional 1 h incubation, PMA (200 nM) was added for 10 min. Cells were then processed according to the C-ELISA protocol. Phosphorylated substrate was measured using an antibody that selectively recognizes the exogenous enzyme substrate expressed only in the transfected cells.