Use Primary Antibodies for Normalization and Reproducibility.

Primary Antibodies for Normalization

Detection of COX IV with rabbit monoclonal primary antibody

Figure 1. Detection of COX IV with rabbit monoclonal primary antibody (P/N 926-42214) is linear across a wide range, even when lysate is heavily loaded. COX IV and actin (rabbit monoclonal; P/N 926-42210) were detected, using IRDye® 800CW Goat anti-Rabbit (P/N 926-32211) and the Odyssey® Imager. This COX IV antibody is an excellent loading control for normalization of low-abundance target proteins. For highly expressed proteins like actin, heavy loading (> 15 µg lysate/lane) may affect linearity of detection.

When performing two-color detection, loading controls and normalization are essential for reliable, precise comparison of protein levels. LI-COR offers five primary antibodies for normalization — antibodies to actin, tubulin, and COX IV — that can be used for two-color normalization when performing multiplex Western blots or for cell-based assay normalization, such as for In-Cell Western™ Assays.

For Western blots, lysate preparation, sample loading, and membrane transfer introduce unavoidable variation. Housekeeping proteins can be used for normalization of protein levels.

  • Normalize and correct for uneven loading, using the intensity of the internal control band
  • Visually compare protein levels with confidence, even if you don't quantify bands
  • Ensure that visually-observed changes in protein levels represent actual change, not artifacts
  • Read about types of internal control proteins to use

When lanes are heavily loaded with lysate (>15 µg/lane), a highly expressed internal control protein may not be an appropriate choice for normalization. For quantitative analysis of a low-abundance target protein, a low-abundance internal control is recommended. COX IV is an appropriate choice.

For In-Cell Western Assays, a second protein target (such as actin, tubulin, COX IV, or GAPDH) can be used for normalization. Abundance of the normalization target must be unaffected by the cell treatments used. Learn more about ICW assay normalization methods.

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