A protocol for recombinant protein quantification by densitometry

This study presents a protocol of image analysis for electroph oresis 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 s ole protein standard for protein quantification in SDS‐PAGE gel images.
Source: MicrobiologyOpen - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research