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Normalize to an Internal Control Protein:

Combined Detection of Total Protein and Phospho-Protein Levels


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.

  • Combine the phospho-antibody with a "total protein" antibody that recognizes the target protein regardless of its phosphorylation status. For example, use mouse anti-total Akt and rabbit anti-phospho-Akt (Fig. 1).
  • This method also corrects for changes in target protein abundance that may be caused by cell treatments.
  • When using two antibodies against the same target, antibody interference may occur (one antibody blocks binding of the other antibody). Controls to rule out antibody inference are very important.
  • To simultaneously detect two target proteins in the ICW assay, you must:

  • Use two primary antibodies that differ in either host species or subclass/isotype
  • AND

  • Use secondary antibodies labeled with different IRDye® fluorophores (700 nm and 800 nm).

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