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.

中文翻译：

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通过光密度法进行重组蛋白定量的方案。

蛋白质纯度通常使用SDS-PAGE检查，其中密度计可用于定量蛋白质条带。在文献中，很少有研究报道使用图像分析技术对SDS-PAGE中的蛋白质进行定量：即使用Stain-Free™技术成像。这项研究提出了电泳凝胶的图像分析协议，该协议允许使用分子量标记作为蛋白质标准品对未知蛋白质进行定量。大肠杆菌由突尼斯巴斯德研究所设计的编码双特异性纳米抗体CH10-12的WK6 / pHEN6在生物反应器中培养，并在28°C下用异丙基β-D-1-硫代半乳糖吡喃糖苷（IPTG）诱导12小时。通过渗透休克提取的周质蛋白通过固定金属亲和色谱法（IMAC）进行纯化。使用ImageJ分析SDS-PAGE凝胶的图像，并获得灰度和未校准光密度的泳道图。蛋白质负载和峰面积线性相关，然后使用半径为250像素的滚动球算法通过背景减法进行最佳图像处理。没有应用亮度和对比度调整。纳米抗体CH10-12的生产是通过补料分批策略获得的，并使用标记中的50 kDa条带作为750 ng重组蛋白的参考进行定量。分子量标记用作SDS-PAGE凝胶图像中蛋白质定量的唯一蛋白质标准品。