![]() The average and the standard deviation of these measurements from 6 repeats were plotted in the graph (E). The image of total protein from staining free gel and the housekeeping protein blotting signals in each lane was measured using Image Lab software following the manufacture’s instruction. For each blot, a stain free image (A) and a chemiluminescent image of a house keeping protein such as β-actin (B), GAPDH (C), or beta-tubulin (D) was taken. Hela cell lysate was loaded at 10, 20, 30, 40, or 50 µg per lane on a Bio-Rad stain free gel, this serial dilution was repeated 6 times in each experiment and the experiment was repeated three times. Stain free total protein measurement reflects the difference in protein load better than the housekeeping proteins blotting signals. These findings raise a potentially serious question on whether using housekeeping genes as an internal standard reference indeed results in the description of “false-positive” differences in our “standard” assay. ![]() Furthermore, those housekeeping proteins were also altered under some drug and experimental treatments and conditions, cell cycle phase, differentiation or proliferation status, and age. However, increasing evidence shows that the housekeeping proteins are subject to change in many biological conditions, such as neuronal diseases, tissues type, as well as under some specific experimental conditions ( figure 1). Therefore, to accurately compare western blotting signals, one must compensate for these non-sample-related variations in signal intensity. The housekeeping proteins are used as reference proteins to normalize the target protein during western blotting analysis. Because the expression levels of specific target proteins are estimated by relative densities, which are based on the assumption that samples are loaded with the same amount of proteins, it is always necessary to control equal protein loading which is often done by re-probing the western blot membrane with an antibody that recognize a housekeeping protein, such as β-actin, β-tubline and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This mini review will focus on (I) the effects of neuronal and non-neuronal diseases, experimental condition, and tissues-specific roles on alteration of housekeeping genes, and (II) alternative internal standards for gene and protein expression analysis.Īnalysis of specific neuronal associated protein expression is often performed by western blotting, a commonly used method to semiquantitatively measure the protein expression of target proteins by specific antibodies. Therefore, these discoveries raise a potential concern regarding whether using a housekeeping protein as an internal standard for target protein analysis is an appropriate practice. Changes of housekeeping genes are also induced by non-neuronal diseases in various tissues. However, recent studies have shown significant variation in some housekeeping genes both at the mRNA and protein levels in various neuropathological events, such as spinal cord injury and Alzheimer's diseases. The most commonly used housekeeping proteins are β-actin, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This practice is based on the belief that such housekeeping genes are considered to be ubiquitously and constitutively expressed in every tissue and produce the minimal essential transcripts necessary for normal cellular function. Housekeeping proteins are used as an internal control for protein loading as well as reference in the western blotting analysis. Study of specific target protein expression is often performed by western blotting, a commonly used method to measure the protein expression in neuroscience research by specific antibodies.
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