Method for CaOx crystals isolation from plant leaves

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.