Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus

Abstract

An ancient medicinal plant known as Orthosiphon stamineus purple variety exhibiting multiple remedial effects by the bioactive compound had been reported. However, no study on its protein separation work was documented. Indeed, two-dimensional (2D) electrophoresis systems have few major issues such as complex time-consuming procedure and poor reproducibility result. Therefore, this proteomic research can be utilized as preliminary platform in identification of protein responsible for the production of bioactive compound and its application as remedies. Different sample require different voltage of isoelectric focusing (IEF) to separate the protein due to its isoelectric point (pI) hence this study was conducted in order to determine the best IEF method producing reproducible protein spots and to determine the protein spot distribution and pattern from 2D proteomics of O. stamineus. Initially, protein extraction utilizing unmodified and modified (1 mL of 80% methanol in 0.1M ammonium acetate, 0.6 mL of each phenol and SDS buffer in 5 % ß-mercaptoethanol, and 0.15mL lysis buffer) phenol extraction with three preliminary washes were performed. The extracted protein was then quantified by Bradford Assay whereas its quality was checked via Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). The modified extraction method resulted in higher spots distribution compared to the unmodified method. In this study, three different IEF method of method 1 (2000V), method 2 (4000V), and method 3 (8000V) were applied before the protein spots pattern and distribution were analysed further. It was found that method 2 was the best IEF method for this plant as 2-fold higher reproducible protein spots were produced. In fact, 15 proteins were expected to be associated with the protein spots obtained that may have high relation with photosynthesis and antioxidant activity. The findings suggested that numerous valuable proteins are expressed in O. stamineus. Hence, the proteins mechanism and properties should be revealed to serve the medical sector in the future.