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使用带荧光检测的成像毛细管等电聚焦和毛细管免疫印迹法对CRM197进行定量分析。

Quantitation of CRM197 using imaged capillary isoelectric focusing with fluorescence detection and capillary Western.

作者信息

Loughney John W, Ha Sha, Rustandi Richard R

机构信息

Biologics and Vaccines, Merck Research Laboratories, Merck and Co., Inc., Kenilworth, NJ, USA.

Biologics and Vaccines, Merck Research Laboratories, Merck and Co., Inc., Kenilworth, NJ, USA.

出版信息

Anal Biochem. 2017 Oct 1;534:19-23. doi: 10.1016/j.ab.2017.06.013. Epub 2017 Jun 27.

Abstract

Maurice is a new instrument that can perform imaged capillary isoelectric focusing (icIEF). The standard detection for icIEF is UV absorbance at 280 nm, which limits its application to high protein concentration samples and non-complex samples. Here we describe an icIEF instrument with fluorescence detection. We demonstrate the advantage of using either icIEF with fluorescence detection or quantitative Western Blot to measure diphtheria toxin mutant CRM197 protein titer in crude cell lysates and purified samples. These two techniques have great potentials to become standard methods to analyze protein titers in crude cell lysate or other complex samples types.

摘要

莫里斯是一种能够进行成像毛细管等电聚焦(icIEF)的新型仪器。icIEF的标准检测方法是280nm处的紫外吸光度,这限制了其在高蛋白浓度样品和非复杂样品中的应用。在此,我们描述了一种具有荧光检测功能的icIEF仪器。我们展示了使用带荧光检测的icIEF或定量蛋白质免疫印迹法来测量粗细胞裂解物和纯化样品中白喉毒素突变体CRM197蛋白滴度的优势。这两种技术极有可能成为分析粗细胞裂解物或其他复杂样品类型中蛋白质滴度的标准方法。

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