<P> In biological data analysis, normalization is an important procedure used to remove both biological and non-biological sources of experimental variability . For accurate, reproducible quantitative analysis of proteins via western blotting, normalization accounts for differences arising from cell culture conditions, inconsistent sample preparation, uneven protein transfer and unequal sample loading across gel lanes . Traditionally for western blots, normalization involves comparing the relative abundance of the protein of interest to that of an unrelated control protein such as a housekeeping protein, to ensure that visually assessed changes in protein levels represent true biological variation and not experimental artifacts . Although Western blot normalization is heading toward total protein normalization instead of using housekeeping proteins, the method is still relevant to genetic studies . </P> <P> The normalization control is a protein that, ideally, is expressed constantly at the same level across different experimental conditions . As such, housekeeping genes and proteins are often used as internal normalization standards in quantitative PCR (qPCR) and western blots . These proteins were designated as housekeeping because they are essential for the maintenance of basic cellular functions and are universally expressed in all cells of an organism . Some examples of classically used housekeeping proteins include ß - Actin, GAPDH, HPRT1, and RPLP1 . Until recently, these proteins were considered to be expressed at a consistent, stable level under normal or pathophysiological conditions in any cell or tissue type . However, recent studies have shown that expression of housekeeping proteins can change across different cell types and biological conditions . It is critical to carefully validate any housekeeping protein chosen as a normalization standard against the sample type and experimental condition . Although normalization reagents (such as antibodies against key proteins) are common and effective, HKP expression can be impacted by experimental conditions . As a result, normalization control must be validated . </P> <P> The detection method in this section is antibody - based and requires a linear relationship between signal intensity and the sample mass or volume to be confirmed for every antigen . Both the target protein and the normalization control should be within the dynamic range of detection . Many housekeeping proteins are expressed at high levels, and are only appropriate for use with highly expressed target proteins . Lower expressing proteins are difficult to detect on the same blot . This is further complicated by the fact that some detection methods, in particular, enhanced chemiluminescence using x-ray film, have a very limited linear range . </P> <P> While antibodies are commercially available, validation techniques may not be consistent and poorly characterized antibodies may yield nonspecific results with low reproducibility . </P>

What is the purpose of a loading control in a western blot