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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...

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Autores principales: Alonso Villela, Susana María, Kraïem, Hazar, Bouhaouala‐Zahar, Balkiss, Bideaux, Carine, Aceves Lara, César Arturo, Fillaudeau, Luc
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
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.
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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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