Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

Abstract Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their i...

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Autores principales:	Narendra Padhan, Junhong Yan, Annegret Boge, Elaine Scrivener, Helgi Birgisson, Agata Zieba, Mats Gullberg, Masood Kamali-Moghaddam, Lena Claesson-Welsh, Ulf Landegren
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5fd5a922af6646128a84ed14eb0a59b9
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spelling	oai:doaj.org-article:5fd5a922af6646128a84ed14eb0a59b92021-12-02T12:30:44ZHighly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation10.1038/s41598-017-01516-72045-2322https://doaj.org/article/5fd5a922af6646128a84ed14eb0a59b92017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01516-7https://doaj.org/toc/2045-2322Abstract Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.Narendra PadhanJunhong YanAnnegret BogeElaine ScrivenerHelgi BirgissonAgata ZiebaMats GullbergMasood Kamali-MoghaddamLena Claesson-WelshUlf LandegrenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Narendra Padhan Junhong Yan Annegret Boge Elaine Scrivener Helgi Birgisson Agata Zieba Mats Gullberg Masood Kamali-Moghaddam Lena Claesson-Welsh Ulf Landegren Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
description	Abstract Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
format	article
author	Narendra Padhan Junhong Yan Annegret Boge Elaine Scrivener Helgi Birgisson Agata Zieba Mats Gullberg Masood Kamali-Moghaddam Lena Claesson-Welsh Ulf Landegren
author_facet	Narendra Padhan Junhong Yan Annegret Boge Elaine Scrivener Helgi Birgisson Agata Zieba Mats Gullberg Masood Kamali-Moghaddam Lena Claesson-Welsh Ulf Landegren
author_sort	Narendra Padhan
title	Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
title_short	Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
title_full	Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
title_fullStr	Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
title_full_unstemmed	Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
title_sort	highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/5fd5a922af6646128a84ed14eb0a59b9
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Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

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