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, 92 (13), 747-51

An Old Method Facing a New Challenge: Re-Visiting Housekeeping Proteins as Internal Reference Control for Neuroscience Research


An Old Method Facing a New Challenge: Re-Visiting Housekeeping Proteins as Internal Reference Control for Neuroscience Research

Rena Li et al. Life Sci.


The 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. Housekeeping proteins are used as an internal control for protein loading as well as reference in the western blotting analysis. 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. The most commonly used housekeeping proteins are β-actin, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). 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. Changes of housekeeping genes are also induced by non-neuronal diseases in various tissues. 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. This minireview will focus on (I) the effects of neuronal and non-neuronal diseases, experimental condition, and tissue-specific roles on alteration of housekeeping genes, and (II) alternative internal standards for gene and protein expression analysis.


Figure 1
Figure 1
Stain free total protein measurement reflects the difference in protein load better than the housekeeping proteins blotting signals. 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. 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. 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. The average and the standard deviation of these measurements from 6 repeats were plotted in the graph (E). The intensity of the protein bands or total protein measurement for the lane loaded with 10 µg of the Hela cell lysate was normalized to 1. The dotted line indicates the quantitative response curve where the relative intensity from the lanes with 50 µg of protein load was expected to be 5.

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