Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase

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Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase. / Thomassen, Martin; Bangsbo, Jens; Hostrup, Morten.

I: Analytical Biochemistry, Bind 666, 115071, 2023.

Publikation: Bidrag til tidsskriftTidsskriftartikelfagfællebedømt

Harvard

Thomassen, M, Bangsbo, J & Hostrup, M 2023, 'Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase', Analytical Biochemistry, bind 666, 115071. https://doi.org/10.1016/j.ab.2023.115071

APA

Thomassen, M., Bangsbo, J., & Hostrup, M. (2023). Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase. Analytical Biochemistry, 666, [115071]. https://doi.org/10.1016/j.ab.2023.115071

Vancouver

Thomassen M, Bangsbo J, Hostrup M. Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase. Analytical Biochemistry. 2023;666. 115071. https://doi.org/10.1016/j.ab.2023.115071

Author

Thomassen, Martin ; Bangsbo, Jens ; Hostrup, Morten. / Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase. I: Analytical Biochemistry. 2023 ; Bind 666.

Bibtex

@article{9acee3bb36a543ad9a9a596ae9479b11,
title = "Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase",
abstract = "Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAβ1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.",
keywords = "Faculty of Science, Western blotting, Biochemical method, Immuno-assay, Muscle physiology, Type-2 diabetes",
author = "Martin Thomassen and Jens Bangsbo and Morten Hostrup",
note = "Copyright {\textcopyright} 2023. Published by Elsevier Inc.",
year = "2023",
doi = "10.1016/j.ab.2023.115071",
language = "English",
volume = "666",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase

AU - Thomassen, Martin

AU - Bangsbo, Jens

AU - Hostrup, Morten

N1 - Copyright © 2023. Published by Elsevier Inc.

PY - 2023

Y1 - 2023

N2 - Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAβ1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

AB - Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAβ1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

KW - Faculty of Science

KW - Western blotting

KW - Biochemical method

KW - Immuno-assay

KW - Muscle physiology

KW - Type-2 diabetes

U2 - 10.1016/j.ab.2023.115071

DO - 10.1016/j.ab.2023.115071

M3 - Journal article

C2 - 36736987

VL - 666

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

M1 - 115071

ER -

ID: 334952209