Fed-batch culture of WK6 strain, induction at 29°C - BR09

5L bioreactor culture of reference strain E. coli WK6. Protein induction promoter when biomass reached 25 g cdw/L and at 29°C for 28 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Characterization of E. coli WK6 strain under in...

Fed-batch culture of NbF12-10 strain, induction at 29°C - BR08

5L bioreactor culture of recombinant strain E. coli NbF12-10. Protein induction promoter when biomass reached 30 g cdw/L and at 29°C for 37 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 and NbF12-10 produ...

Fed-batch culture of CH10-12 strain, induction at 32°C - BR07

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 30 g cdw/L and at 32°C for 35 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 and NbF12-10 produ...

Fed-batch culture of CH10-12 strain, induction at 29°C - BR06

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 30 g cdw/L and at 29°C for 38 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 and NbF12-10 produ...

Fed-batch culture of CH10-12 strain, induction at 37°C - BR05

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 25 g cdw/L and at 37°C for 6 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 production at diff...

Fed-batch culture of CH10-12 strain, induction at 33°C - BR04

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 25 g cdw/L and at 33°C for 6 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 production at diff...

Fed-batch culture of CH10-12 strain, induction at 30°C - BR03

5L bioreactor culture of recombinant strain E. coli CH10-12. Protein induction promoter when biomass reached 25 g cdw/L and at 30°C for 10 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 production at diff...

Fed-batch culture of CH10-12 strain, induction at 28°C - BR02

5L bioreactor culture of recombinant strain E. coli CH10-12 in a batch/fed-batch strategy at 37°C (BR02). Protein induction promoted when biomass reached 25 g cdw/L and at 28°C for 12 h

Submitter: Susana Alonso Villela

Assay type: Cultivation Experiment

Technology type: Fed-batch

Investigation: Effect of the induction temperature in nanobody...

Study: Recombinant nanobody CH10-12 production at diff...

A protocol for recombinant protein quantification by densitometry

Abstract (Expand)

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.

Authors: Susana María Alonso Villela, Hazar Kraïem, Balkiss Bouhaouala‐Zahar, Carine Bideaux, César Arturo Aceves Lara, Luc Fillaudeau

Date Published: 1st Jun 2020

Publication Type: Journal

MFCS BioPAT - BR04

5L bioreactor of E. coli CH10-12 in a batch/fed-batch strategy at 37°C, induction promoted for 6h at 33°C MFCS BioPAT Sartorius

MFCS BioPAT - BR03

5L bioreactor of E. coli CH10-12 in a batch/fed-batch strategy at 37°C, induction promoted for 10h at 30°C MFCS BioPAT Sartorius

MFCS BioPAT - BR02

5L bioreactor of E. coli CH10-12 in a batch/fed-batch strategy at 37°C, induction promoted for 12h at 28°C MFCS BioPAT Sartorius

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