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Evaluation of antibodies for western blot analysis of frataxin protein isoforms

Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-lengt...

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Autores principales: Weng, Liwei, Wang, Qingqing, Yu, Sixiang, Yang, Xiaolu, Lynch, David R., Mesaros, Clementina, Blair, Ian A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829029/
https://www.ncbi.nlm.nih.gov/pubmed/31279523
http://dx.doi.org/10.1016/j.jim.2019.07.001
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author Weng, Liwei
Wang, Qingqing
Yu, Sixiang
Yang, Xiaolu
Lynch, David R.
Mesaros, Clementina
Blair, Ian A.
author_facet Weng, Liwei
Wang, Qingqing
Yu, Sixiang
Yang, Xiaolu
Lynch, David R.
Mesaros, Clementina
Blair, Ian A.
author_sort Weng, Liwei
collection PubMed
description Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-length frataxin (1−210) protein. Full length frataxin translocates to the mitochondria, leading to formation of mature frataxin (81–210) formed by cleavage of the mitochondrial targeting sequence at K-80 of the full-length protein. There are currently no approved treatments for FRDA, although experimental approaches involving up-regulation or replacement of mature frataxin protein through numerous approaches are being tested. Many of the pre-clinical studies of these experimental approaches are conducted in mouse and monkey models as well as in human cell lines. Consequently, well-validated antibodies are required for use in western blot analysis to determine whether levels of various forms of frataxin have been increased. Here we examined the specificity of five commercially available anti-frataxin antibodies and determined whether they detect mature frataxin in mouse heart tissue. Four protein standards of monkey, human, and mouse frataxin as well as mouse heart tissue were examined using polyacrylamide gel electrophoresis (PAGE) in combination with western blot analysis. One antibody failed to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue. Three of the antibodies detected a protein in mouse heart tissue that ran slightly faster on PAGE (at 23.4 kDa) to that predicted for full-length frataxin (23.9 kDa). One antibody detected all four frataxin standards as well as endogenous mouse mature frataxin in mouse tissue. Significantly, this antibody, which will be useful for monitoring mature frataxin levels in monkey, human, and mouse tissues, did not detect a protein in mouse heart tissue at 23.4 kDa. Therefore, antibodies detecting the immunoreactive protein at 23.4 kDa could be misleading when testing for the up-regulation of frataxin in animal models.
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spelling pubmed-68290292019-11-05 Evaluation of antibodies for western blot analysis of frataxin protein isoforms Weng, Liwei Wang, Qingqing Yu, Sixiang Yang, Xiaolu Lynch, David R. Mesaros, Clementina Blair, Ian A. J Immunol Methods Article Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-length frataxin (1−210) protein. Full length frataxin translocates to the mitochondria, leading to formation of mature frataxin (81–210) formed by cleavage of the mitochondrial targeting sequence at K-80 of the full-length protein. There are currently no approved treatments for FRDA, although experimental approaches involving up-regulation or replacement of mature frataxin protein through numerous approaches are being tested. Many of the pre-clinical studies of these experimental approaches are conducted in mouse and monkey models as well as in human cell lines. Consequently, well-validated antibodies are required for use in western blot analysis to determine whether levels of various forms of frataxin have been increased. Here we examined the specificity of five commercially available anti-frataxin antibodies and determined whether they detect mature frataxin in mouse heart tissue. Four protein standards of monkey, human, and mouse frataxin as well as mouse heart tissue were examined using polyacrylamide gel electrophoresis (PAGE) in combination with western blot analysis. One antibody failed to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue. Three of the antibodies detected a protein in mouse heart tissue that ran slightly faster on PAGE (at 23.4 kDa) to that predicted for full-length frataxin (23.9 kDa). One antibody detected all four frataxin standards as well as endogenous mouse mature frataxin in mouse tissue. Significantly, this antibody, which will be useful for monitoring mature frataxin levels in monkey, human, and mouse tissues, did not detect a protein in mouse heart tissue at 23.4 kDa. Therefore, antibodies detecting the immunoreactive protein at 23.4 kDa could be misleading when testing for the up-regulation of frataxin in animal models. 2019-07-04 2019-11 /pmc/articles/PMC6829029/ /pubmed/31279523 http://dx.doi.org/10.1016/j.jim.2019.07.001 Text en https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Weng, Liwei
Wang, Qingqing
Yu, Sixiang
Yang, Xiaolu
Lynch, David R.
Mesaros, Clementina
Blair, Ian A.
Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title_full Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title_fullStr Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title_full_unstemmed Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title_short Evaluation of antibodies for western blot analysis of frataxin protein isoforms
title_sort evaluation of antibodies for western blot analysis of frataxin protein isoforms
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829029/
https://www.ncbi.nlm.nih.gov/pubmed/31279523
http://dx.doi.org/10.1016/j.jim.2019.07.001
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