![]() Thus, to detect the target protein of interest, large amounts of cell lysate may need to be loaded, resulting in overloading of HKPs, yielding oversaturated reference bands, out of their linear range. ![]() Unfortunately, housekeeping proteins are usually highly expressed, whereas target proteins are often expressed only in low abundance. Reliable assessment of the changes in target protein expression levels requires the measurement of both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Typically, a "housekeeping protein" (HKP) such as β-actin, β-tubulin, or GAPDH is used as a loading control, with the assumption that the expression levels of these proteins remains constant. Proper western blot normalization is required to show that the changes in band intensities correlate to the biological changes in your samples.Ĭonventional loading controls rely on consistent levels of a reference protein in each sample. Western blot normalization allows you to faithfully compare changes in protein expression by establishing the baseline needed to correct against common errors such as inconsistent sample preparation, pipetting error, and uneven protein transfer. Common blot-to-blot variations are accurately assessed by an internal loading control. ![]()
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