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Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays
BACKGROUND: Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many p...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339431/ https://www.ncbi.nlm.nih.gov/pubmed/25527077 http://dx.doi.org/10.1186/s12865-014-0062-z |
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author | Luo, Yuqian Hara, Takeshi Ishido, Yuko Yoshihara, Aya Oda, Kenzaburo Makino, Masahiko Ishii, Norihisa Hiroi, Naoki Suzuki, Koichi |
author_facet | Luo, Yuqian Hara, Takeshi Ishido, Yuko Yoshihara, Aya Oda, Kenzaburo Makino, Masahiko Ishii, Norihisa Hiroi, Naoki Suzuki, Koichi |
author_sort | Luo, Yuqian |
collection | PubMed |
description | BACKGROUND: Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions. RESULTS: We have developed a simple, rapid and cost-effective method to prepare highly purified nuclear proteins from a small number of both suspended and adherent cultured cells that yields nuclear proteins comparable to those prepared by a standard large-scale method. The efficiency of the method was demonstrated by using EMSA to show the successful detection, in multilple concurrent samples, of NF-κB activation upon tetradecanoyl phorbol acetate (TPA) stimulation. CONCLUSIONS: This method requires only a small number of cells and no specialized equipment. The steps have been simplified, resulting in a short processing time, which allows researchers to process multiple samples simultaneously and quickly. This method is especially optimized for use in EMSA, and may be useful for other applications that include proteomic analysis. |
format | Online Article Text |
id | pubmed-4339431 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-43394312015-02-26 Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays Luo, Yuqian Hara, Takeshi Ishido, Yuko Yoshihara, Aya Oda, Kenzaburo Makino, Masahiko Ishii, Norihisa Hiroi, Naoki Suzuki, Koichi BMC Immunol Methodology Article BACKGROUND: Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions. RESULTS: We have developed a simple, rapid and cost-effective method to prepare highly purified nuclear proteins from a small number of both suspended and adherent cultured cells that yields nuclear proteins comparable to those prepared by a standard large-scale method. The efficiency of the method was demonstrated by using EMSA to show the successful detection, in multilple concurrent samples, of NF-κB activation upon tetradecanoyl phorbol acetate (TPA) stimulation. CONCLUSIONS: This method requires only a small number of cells and no specialized equipment. The steps have been simplified, resulting in a short processing time, which allows researchers to process multiple samples simultaneously and quickly. This method is especially optimized for use in EMSA, and may be useful for other applications that include proteomic analysis. BioMed Central 2014-12-20 /pmc/articles/PMC4339431/ /pubmed/25527077 http://dx.doi.org/10.1186/s12865-014-0062-z Text en © Luo et al.; licensee BioMed Central. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Luo, Yuqian Hara, Takeshi Ishido, Yuko Yoshihara, Aya Oda, Kenzaburo Makino, Masahiko Ishii, Norihisa Hiroi, Naoki Suzuki, Koichi Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title | Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title_full | Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title_fullStr | Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title_full_unstemmed | Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title_short | Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
title_sort | rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339431/ https://www.ncbi.nlm.nih.gov/pubmed/25527077 http://dx.doi.org/10.1186/s12865-014-0062-z |
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