Housekeeping proteins: A preliminary study illustrating some limitations as useful references in protein expression studies

Abstract

A primary objective of many protein expression studies is to define expression patterns that can distinguish between normal and diseased states, enabling a better understanding of molecular events associated with disease development and progression and ultimately potentially finding novel markers or therapeutic targets. Exploration and confirmation of many proteins is often done using Western blotting with normalization against "housekeeping proteins", such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, or beta-tubulin, to correct for protein loading and factors, such as transfer efficiency. Increasingly, in studies examining gene transcript levels, it has been shown that some of the commonly used housekeeping genes may be unsuitable due to the influence of various physiological and pathological factors on their expression. This has not been examined to any great extent for proteins, however. This study examines the degree of variability of three commonly used "housekeeping" proteins (GAPDH, beta-actin, and beta-tubulin) together with class I beta-tubulin, with comparisons being made between a number of different established renal cancer cell lines, matched pairs of renal tumor and normal kidney lysates as well as nine different human tissues and highlights some of the problems encountered.

Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinoma

“…Concentrations of mRNA are expressed relative to the level of b-actin. Even though Ferugson et al [15] showed the higher expression of b-actin in RCC, it was marginal. Statistical significance of differences between mean values was calculated using the paired t-test and a p,0.05 (two-tailed) was considered to be significant.…”

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-κB and sterol regulatory element-binding protein-1 in adipocytes

“…GLUT4 content was expressed in arbitrary units per microgram of applied protein, considering the mean of control animals 100%. Protein-loaded content was confirmed by analyzing posttransferring Coomassie-stained gels (Ferguson et al 2005). …”

The mGluR2/3 Agonist LY379268 Blocks the Effects of GLT-1 Upregulation on Prepulse Inhibition of the Startle Reflex in Adult Rats

“…A standard curve with 2, 4, 6, 8, and 10 mg of bovine serum albumin (A4503, Sigma Chemicals, St Louis, MO, USA) was drawn for each dosing run. As housekeeping proteins (such as a-actin and b-tubulins) are sensitive to experimental treatments (particularly pharmacologic treatments) and to diverse physiological conditions and have therefore some limitations as internal standard (Ferguson et al, 2005), 3-6 measurements were made for each brain region of each animal; to minimize procedural variables, homogenates from treated and control animals LY379268 blocks CEF-induced PPI impairment M Bellesi and F Conti were loaded onto the same gel (Bragina et al, 2006). For a semi-quantitative analysis, we drew standard curves of increasing concentration of total protein from controls (n ¼ 4) to define a linear range for immunoblot densitometric analysis (Bragina et al, 2006).…”