Primary Antibodies for Normalization by LI-COR Biosciences

When performing two-color detection, loading controls and normalization are essential for reliable, precise comparison of protein levels. 

LI-COR offers nine primary antibodies for normalization – antibodies to actin, COX IV, GAPDH, histone h3, tubulin, and vinculin – 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, COXIV, or GAPDH) can be used for normalization. Abundance of the normalization target must be unaffected by the cell treatments used.