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A protocol for recombinant protein quantification by densitometry
The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294310/ https://www.ncbi.nlm.nih.gov/pubmed/32255275 http://dx.doi.org/10.1002/mbo3.1027 |
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author | Alonso Villela, Susana María Kraïem, Hazar Bouhaouala‐Zahar, Balkiss Bideaux, Carine Aceves Lara, César Arturo Fillaudeau, Luc |
author_facet | Alonso Villela, Susana María Kraïem, Hazar Bouhaouala‐Zahar, Balkiss Bideaux, Carine Aceves Lara, César Arturo Fillaudeau, Luc |
author_sort | Alonso Villela, Susana María |
collection | PubMed |
description | The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images. |
format | Online Article Text |
id | pubmed-7294310 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-72943102020-06-15 A protocol for recombinant protein quantification by densitometry Alonso Villela, Susana María Kraïem, Hazar Bouhaouala‐Zahar, Balkiss Bideaux, Carine Aceves Lara, César Arturo Fillaudeau, Luc Microbiologyopen Original Articles The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images. John Wiley and Sons Inc. 2020-04-07 /pmc/articles/PMC7294310/ /pubmed/32255275 http://dx.doi.org/10.1002/mbo3.1027 Text en © 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Articles Alonso Villela, Susana María Kraïem, Hazar Bouhaouala‐Zahar, Balkiss Bideaux, Carine Aceves Lara, César Arturo Fillaudeau, Luc A protocol for recombinant protein quantification by densitometry |
title | A protocol for recombinant protein quantification by densitometry |
title_full | A protocol for recombinant protein quantification by densitometry |
title_fullStr | A protocol for recombinant protein quantification by densitometry |
title_full_unstemmed | A protocol for recombinant protein quantification by densitometry |
title_short | A protocol for recombinant protein quantification by densitometry |
title_sort | protocol for recombinant protein quantification by densitometry |
topic | Original Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294310/ https://www.ncbi.nlm.nih.gov/pubmed/32255275 http://dx.doi.org/10.1002/mbo3.1027 |
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