GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

Abstract

Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate‐intensity exercise in human skeletal muscle

“…Given that we have observed previously that glyceraldehyde‐3‐dehydrogenase, β‐actin and α‐tubulin are not suitable as loading controls in this data set (Vigelsø et al . a ), the intensities of the bands of interest were normalized to the total Stain‐Free fluorescence (total protein).…”

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

“…However, a body of evidence has gathered showing that HKG expression may in fact vary between different tissues or cell lines and change in response to pathology, treatment, or altered environmental conditions (Dheda et al 2005). Moreover, glyceraldehade-3-phosphate dehydrogenase (GAPDH), the most frequently used normalizer, has been demonstrated to increase over 40-fold in severe sepsis (Cummings et al 2014) but decrease with ageing (Vigelsø et al 2015). Concerning cancer, GAPDH confers growth advantage and hence is frequently up-regulated in tumor cells (reviewed in Guo et al 2013; Ramos et al 2015).…”

Β‐actin does not show the characteristics of a reference protein in human cortex

“…Our data from BA 9 from 194 subjects, some of which had a psychiatric disorder, does display some of the characteristics of a reference protein in that levels of β‐actin did not vary with gender, age, PMI, CNS pH, CNS weight, diagnoses, DI or suicide completion. However, these findings may be CNS specific as it has been reported that in human muscle levels of β‐actin decrease with age .…”

“…In considering alternative approaches to the use of reference proteins, others have suggested that using carefully measured total protein loaded onto a gel , total protein measured in a gel using stain free technologies or the intensity of a Ponceau S staining of transferred proteins as alternative normalisation measurements. Our data suggests normalising to Ponceau S staining would introduce less variability than normalising to β‐actin but our QCC data suggest that the variability introduced by using transferred protein levels would still be significant.…”

“…If such data is presented alongside appropriate quality control data, it would be possible to determine if changes in proteins with variables such as diagnoses were greater than the methodological variance. In addition to an analysis of non‐derived Western blot data, it would be valuable to show that total protein in stain free gels or integrated measures of Ponceau S staining did not vary with diagnoses. Alternatively, other more stable measures of protein levels, such as staining with Sypro ® ‐Ruby , Amido Black or post staining with Coomassie R‐250 may be more useful as they provide more stable read‐outs when measuring protein loading of gels.…”