Figure 1. In-Cell Western analysis of Akt phosphorylation. A) MCF-7 breast cancer cells were treated with recombinant human IGF-1. After fixation and permeabilization in microplate wells, phospho-Akt (S473) and total Akt protein levels were detected. B) Levels of phospho-Akt, normalized to total Akt protein levels in the same well, were quantified by analysis of fluorescence intensities. Phospho-Akt and total Akt were also detected by Western blot (inset). R&D Systems Phospho-Akt (S473) Cell-Based Infrared ELISA kit (KCBIR887) was used.
It may be possible to use the target protein as its own internal control for detection of a phospho-protein. Total levels of the target protein, and extent of phosphorylation of the target, are measured simultaneously.
To simultaneously detect two target proteins in the ICW assay, you must: