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

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Standard

GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. / Vigelsø Hansen, Andreas; Dybboe, Rie; Hansen, Christina Neigaard; Dela, Flemming; Helge, Jørn Wulff; Guadalupe Grau, Amelia.

I: Journal of Applied Physiology, Bind 118, Nr. 3, 2015, s. 386-394.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Vigelsø Hansen, A, Dybboe, R, Hansen, CN, Dela, F, Helge, JW & Guadalupe Grau, A 2015, 'GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle', Journal of Applied Physiology, bind 118, nr. 3, s. 386-394. https://doi.org/10.1152/japplphysiol.00840.2014

APA

Vigelsø Hansen, A., Dybboe, R., Hansen, C. N., Dela, F., Helge, J. W., & Guadalupe Grau, A. (2015). GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. Journal of Applied Physiology, 118(3), 386-394. https://doi.org/10.1152/japplphysiol.00840.2014

Vancouver

Vigelsø Hansen A, Dybboe R, Hansen CN, Dela F, Helge JW, Guadalupe Grau A. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. Journal of Applied Physiology. 2015;118(3):386-394. https://doi.org/10.1152/japplphysiol.00840.2014

Author

Vigelsø Hansen, Andreas ; Dybboe, Rie ; Hansen, Christina Neigaard ; Dela, Flemming ; Helge, Jørn Wulff ; Guadalupe Grau, Amelia. / GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. I: Journal of Applied Physiology. 2015 ; Bind 118, Nr. 3. s. 386-394.

Bibtex

@article{02352d2e2692404c94b50f53db4d10ce,
title = "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.",
author = "{Vigels{\o} Hansen}, Andreas and Rie Dybboe and Hansen, {Christina Neigaard} and Flemming Dela and Helge, {J{\o}rn Wulff} and {Guadalupe Grau}, Amelia",
note = "CURIS 2015 NEXS 057",
year = "2015",
doi = "10.1152/japplphysiol.00840.2014",
language = "English",
volume = "118",
pages = "386--394",
journal = "Journal of Applied Physiology",
issn = "8750-7587",
publisher = "American Physiological Society",
number = "3",

}

RIS

TY - JOUR

T1 - GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

AU - Vigelsø Hansen, Andreas

AU - Dybboe, Rie

AU - Hansen, Christina Neigaard

AU - Dela, Flemming

AU - Helge, Jørn Wulff

AU - Guadalupe Grau, Amelia

N1 - CURIS 2015 NEXS 057

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

U2 - 10.1152/japplphysiol.00840.2014

DO - 10.1152/japplphysiol.00840.2014

M3 - Journal article

C2 - 25429098

VL - 118

SP - 386

EP - 394

JO - Journal of Applied Physiology

JF - Journal of Applied Physiology

SN - 8750-7587

IS - 3

ER -

ID: 131165013