[1][2] The overall goal of normalization is to minimize effects arising from variations in experimental errors, such as inconsistent sample preparation, unequal sample loading across gel lanes, or uneven protein transfer, which can compromise the conclusions that can be obtained from Western blot data.
[5] Housekeeping genes and proteins, including β-Actin, GAPDH, HPRT1, and RPLP1, are often used as internal controls in western blots because they are thought to be expressed constitutively, at the same levels, across experiments.
[1][2][6][7] However, recent studies have shown that expression of housekeeping proteins (HKPs) can change across different cell types and biological conditions.
[1][8][9][10] Therefore, scientific publishers and funding agencies now require that normalization controls be previously validated for each experiment to ensure reproducibility and accuracy of the results.
[8][9][10] When using fluorescent antibodies to image proteins in western blots, normalization requires that the user define the upper and lower limits of quantitation and characterize the linear relationship between signal intensity and the sample mass volume for each antigen.
[1] Both the target protein and the normalization control need to fluoresce within the dynamic range of detection.
[17][18][19][20] Ponceau S is a negatively charged reversible dye that stains proteins a reddish pink color and is removed easily by washing in water.
[5] A linear range of up to 140 μg is reported for Ponceau S with poor reproducibility due to its highly time-dependent staining intensity and low signal-to-noise ratio.
[22] They are permanent, photostable stains that can be visualized with a standard UV or blue-light transilluminator or a laser scan.