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Related Experiment Video

Updated: Dec 24, 2025

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB
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A protocol for recombinant protein quantification by densitometry.

Susana María Alonso Villela1, Hazar Kraïem2, Balkiss Bouhaouala-Zahar2,3

  • 1TBI, CNRS, INRAE, INSA, Université de Toulouse, Toulouse, France.

Microbiologyopen
|April 8, 2020
PubMed
Summary

This study introduces a novel image analysis protocol for quantifying proteins in SDS-PAGE gels using molecular weight markers as standards. This method enables accurate protein measurement from electrophoresis images, enhancing bioprocess monitoring.

Keywords:
SDS-PAGEdensitometryimage analysisnanobody productionprotein quantitation

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Area of Science:

  • Biotechnology
  • Biochemistry
  • Molecular Biology

Background:

  • Protein quantification is crucial for biopharmaceutical development and quality control.
  • Traditional methods like densitometry on SDS-PAGE gels have limitations.
  • Image analysis of stained or Stain-Free™ gels offers potential for improved quantification.

Purpose of the Study:

  • To develop and validate an image analysis protocol for quantifying proteins in SDS-PAGE gels.
  • To utilize molecular weight markers as protein standards for accurate quantification.
  • To establish a reliable method for assessing recombinant protein production.

Main Methods:

  • Cultured Escherichia coli expressing bispecific nanobody CH10-12.
  • Purified periplasmic proteins using immobilized metal affinity chromatography (IMAC).
  • Analyzed SDS-PAGE gel images using ImageJ software with background subtraction (rolling ball algorithm).

Main Results:

  • Established a linear correlation between protein load and peak area in gel images.
  • Quantified nanobody CH10-12 production at 750 ng using a 50 kDa marker band as reference.
  • Demonstrated the efficacy of molecular weight markers as sole protein standards for quantification.

Conclusions:

  • The developed image analysis protocol provides a reliable method for protein quantification in SDS-PAGE.
  • This technique enhances the assessment of recombinant protein production in bioprocesses.
  • Image analysis of electrophoresis gels offers a valuable alternative for protein quantitation.