Separation of Bioactive Small Molecules, Peptide from Natural Products and Proteins from Pathogenic Microbe by Rotofor-based Preparative Isoelectric Focusing (IEF) Method

The primary purpose of purifying the bioactive small molecules is obtaining enough amount of specific molecules for their downstream applications. There are various methods available for serving this purpose. However, not all can afford for cost-effective purification methods, especially for complex biological samples. Here we demonstrate, the Rotofor based preparative IEF separation which shows the high possibility of separating and enriching desired molecules, including small molecules and peptides from the complex plant extract. This method is well-suited for use at any stage of a purification scheme and has been used for complex biological samples quantification and characterization. As a proof of concept, we fractionated Gymnema sylvestre plant extract and isolated a family of terpenoid saponin molecules at the acidic end and small peptide molecule near the basic end. To our knowledge, this is a first report that Rotofor can be used to fractionate bioactive small molecules from natural product sources. We also showed effective microbial protein separation using Candida albicans fungus as a model system. The Rotofor based IEF has appealing features such as low cost, simplicity, high application potential and can be performed in any laboratory with the instrument setup.