R&D Systems now offers two new Cell-Based ELISA Kits, a Human/Mouse/Rat Phospho-Akt (T308) Kit (Catalog # KCB8871) and a Human/Mouse Phospho-RPS6 (S235/S236) Kit (Catalog # KCB3918 ). These fluorescence-based immunoassays use fixed, permeabilized cells to simultaneously measure the specified phosphorylated form of either Akt or RPS6 and the respective total protein in the same microplate well. Normalization of the fluorescent signal derived from the phosphorylated protein to that of the total protein corrects for well-to-well variations such as differences in cell number. Cell-Based ELISAs require no specialized equipment or lysate preparation, and can be run with as few as 10,000 cells per well, making them an excellent alternative to Western blot.

Detection of Serum-induced RPS6 Phosphorylation in Mouse Embryonic Fibroblasts. Balb/3T3 mouse embryonic fibroblasts were cultured in 96-well plates and treated with 20% fetal bovine serum for the indicated times. After fixation of the cells, the level of RPS6 phosphorylation on S235/S236 was determined using the Human/Mouse Phospho-RPS6 (S235/S236) Cell-Based ELISA (Catalog # KCB3918) and normalized to total RPS6 detected in the same microplate wells (bar graph). Values represent the mean ± the range of duplicate determinations. The results of the Cell-Based ELISA are consistent with the amounts of phosphorylated RPS6 detected by Western blot analysis (inset).