Cerritos-Castro Ivan T, Patrón-Soberano Araceli, Barba de la Rosa Ana P
IPICYT, Instituto Potosino de Investigación Científica y Tecnológica A.C., San Luis Potosí, San Luis Potosí, 78216 México.
MethodsX. 2022 Jul 28;9:101798. doi: 10.1016/j.mex.2022.101798. eCollection 2022.
Although calcium oxalate (CaOx) crystals are present in many plants they are poorly studied. A possible limitation is the lack of methods for CaOx crystals isolation at high concentration and high purity, which is required for the analysis of their associated biomolecules such as proteins. To our knowledge, there are only four works that have isolated proteins from CaOx crystals. Those methods basically consist of grinding the plant material, filtration steps, enzymatic digestions, and density-based separation. However, they lack of steps to evaluate the quality and purity of the isolated crystals. Likewise, those works do not evaluate whether the crystals obtained carry contaminating proteins. In the present work a detailed method for CaOx crystals isolation from amaranth leaves is described, which can be used to isolate crystals from other plant leaves. The present method is based on previous works with the addition of cleaning steps to removal contaminating protein, separation of crystals by size, and microscopic monitoring to validate the purification efficiency. Main steps for CaOx crystals isolation:•Plant leaves are ground and several washing steps, including enzymatic digestions and centrifugation, are carried out to remove cellular debris and contaminating proteins.•CaOx crystals are enriched by centrifugation in sodium polytungstate.•The different forms of crystals are separated by filtration.
尽管草酸钙（CaOx）晶体存在于许多植物中，但对它们的研究却很少。一个可能的限制是缺乏高浓度和高纯度分离CaOx晶体的方法，而分析其相关生物分子（如蛋白质）需要这种方法。据我们所知，仅有四项研究从CaOx晶体中分离出了蛋白质。这些方法主要包括研磨植物材料、过滤步骤、酶消化以及基于密度的分离。然而，它们缺乏评估分离出的晶体质量和纯度的步骤。同样，这些研究也没有评估所获得的晶体是否携带污染性蛋白质。在本研究中，描述了一种从苋菜叶中分离CaOx晶体的详细方法，该方法可用于从其他植物叶片中分离晶体。本方法基于先前的研究，并增加了去除污染性蛋白质的清洗步骤、按大小分离晶体以及通过显微镜监测来验证纯化效率。CaOx晶体分离的主要步骤：
• 将植物叶片研磨，并进行包括酶消化和离心在内的多个清洗步骤，以去除细胞碎片和污染性蛋白质。
• 通过在多聚钨酸钠中离心来富集CaOx晶体。
• 通过过滤分离不同形态的晶体。
