Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.

<h4>Background</h4>Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported t...

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Autores principales:	Wei Cun See Too, Mun Teng Wong, Ling Ling Few, Manfred Konrad
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/95288dc810b84d8daa3a664c3698d074
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spelling	oai:doaj.org-article:95288dc810b84d8daa3a664c3698d0742021-11-18T06:34:43ZHighly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.1932-620310.1371/journal.pone.0012999https://doaj.org/article/95288dc810b84d8daa3a664c3698d0742010-09-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20886003/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.<h4>Methodology/principal findings</h4>In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.<h4>Conclusion/significance</h4>Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.Wei Cun See TooMun Teng WongLing Ling FewManfred KonradPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 9, p e12999 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Wei Cun See Too Mun Teng Wong Ling Ling Few Manfred Konrad Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
description	<h4>Background</h4>Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.<h4>Methodology/principal findings</h4>In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.<h4>Conclusion/significance</h4>Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.
format	article
author	Wei Cun See Too Mun Teng Wong Ling Ling Few Manfred Konrad
author_facet	Wei Cun See Too Mun Teng Wong Ling Ling Few Manfred Konrad
author_sort	Wei Cun See Too
title	Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
title_short	Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
title_full	Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
title_fullStr	Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
title_full_unstemmed	Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
title_sort	highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/95288dc810b84d8daa3a664c3698d074
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Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.

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