Figure 1. In-Cell Western Assay with CellTag 700 Stain in EGF-stimulated A431 Cells. EGF-stimulated A431 cells were fixed and permeabilized. Phosphorylated EGFR was measured using rabbit anti-phospho-EGFR primary antibody followed by detection with IRDye 800CW Goat anti-Rabbit secondary antibody (P/N 926-32211). CellTag 700 Stain was used for normalization to cell number.
The data demonstrate phosphorylated levels of EGFR increase with treatment of EGF. The plate was scanned on Odyssey® Classic Infrared Imaging System (Resolution: 169 µm; Quality: medium; Focus offset: 4.0 mm; Intensity: 5 for both channels).
The In-Cell Western Assay is an immunocytochemical assay that uses near-infrared fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting, and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. By using the CellTag 700 Stain ICW Kits, the cost per well is reduced to a fraction of the cost of typical screening methods.
CellTag 700 Stain is a near-infrared fluorescent, non-specific cell stain that provides accurate normalization to cell number for In-Cell Western applications. The stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers. CellTag 700 Stain is applied to the cells during incubation with IRDye® 800CW secondary antibody, and enables accurate measurement of target protein levels with much higher throughput than Western blotting.