Modified Western blotting for insulin and other diabetes-associated peptide hormones

Abstract Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western...

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Autores principales:	Naoyuki Okita, Yoshikazu Higami, Fumio Fukai, Masaki Kobayashi, Miku Mitarai, Takao Sekiya, Takashi Sasaki
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/dbc324fac75a4f98a85a3463a609a9d1
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spelling	oai:doaj.org-article:dbc324fac75a4f98a85a3463a609a9d12021-12-02T12:32:49ZModified Western blotting for insulin and other diabetes-associated peptide hormones10.1038/s41598-017-04456-42045-2322https://doaj.org/article/dbc324fac75a4f98a85a3463a609a9d12017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-04456-4https://doaj.org/toc/2045-2322Abstract Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.Naoyuki OkitaYoshikazu HigamiFumio FukaiMasaki KobayashiMiku MitaraiTakao SekiyaTakashi SasakiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Naoyuki Okita Yoshikazu Higami Fumio Fukai Masaki Kobayashi Miku Mitarai Takao Sekiya Takashi Sasaki Modified Western blotting for insulin and other diabetes-associated peptide hormones
description	Abstract Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.
format	article
author	Naoyuki Okita Yoshikazu Higami Fumio Fukai Masaki Kobayashi Miku Mitarai Takao Sekiya Takashi Sasaki
author_facet	Naoyuki Okita Yoshikazu Higami Fumio Fukai Masaki Kobayashi Miku Mitarai Takao Sekiya Takashi Sasaki
author_sort	Naoyuki Okita
title	Modified Western blotting for insulin and other diabetes-associated peptide hormones
title_short	Modified Western blotting for insulin and other diabetes-associated peptide hormones
title_full	Modified Western blotting for insulin and other diabetes-associated peptide hormones
title_fullStr	Modified Western blotting for insulin and other diabetes-associated peptide hormones
title_full_unstemmed	Modified Western blotting for insulin and other diabetes-associated peptide hormones
title_sort	modified western blotting for insulin and other diabetes-associated peptide hormones
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/dbc324fac75a4f98a85a3463a609a9d1
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_version_	1718394010348814336

Modified Western blotting for insulin and other diabetes-associated peptide hormones

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