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.
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.