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Normalization to an Internal Control Protein:

Housekeeping Protein


Figure 1: Staurosporine-induced Caspase-3 cleavage in non-adherent cells. Jurkat human acute T-cell leukemia cells were cultured in 96-well plates and treated with the indicated amounts of staurosporine for 4 h. After fixation of cells in the wells, the amount of Cleaved Caspase-3 (Asp175) was determined using the R&D Systems Human Cleaved Caspase-3 (Asp175) Cell-Based Infrared ELISA kit (KCBIR835). Signals were normalized to GAPDH staining in each well. Values represent the mean ± the range of duplicate determinations. Analysis of Cleaved Caspase-3 (Asp175) and GAPDH by Western blotting, using the antibodies supplied in this kit, is also shown (inset).

A second protein target (such as actin, tubulin, COXIV, or GAPDH) can be used for normalization (Fig. 1). Abundance of the normalization target must be unaffected by the cell treatments used.

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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