A Simple Sonication Method to Isolate the Chloroplast Lumen in Arabidopsis thaliana.

Jingfang Hao, Alizée Malnoë
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Abstract

The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane-associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.

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拟南芥叶绿体管腔的简单超声分离方法研究。
叶绿体管腔包含至少80种蛋白质,其功能和调控尚未完全了解。分离叶绿体管腔可以表征管腔蛋白。可以通过几种方法分离管腔,通过使用叶达压或超声破坏类囊体,或通过使用洗涤剂溶解类囊体。在这里,我们提出了一个简单的程序,分离类囊体腔声波利用植物拟南芥叶片。步骤如下:从叶绿体中分离出类囊体,从基质中去除松散相关的类囊体表面蛋白,超声和离心后在上清中收集管腔部分。与其他方法相比,该方法易于实施,节省时间、植物材料和成本。获得高数量和高纯度的管腔蛋白;然而,一些基质膜相关蛋白被释放到管腔部分,因此如果需要,可以通过降低超声功率和/或时间进一步调整该方法。
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