Journal of Medicinal Plants Research Vol. 5(16), pp. 3777-3780, 18 August, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals Full Length Research Paper Optimization of two-dimensional gel electrophoresis protocols for Boesenbergia rotunda in vitro suspension culture Tan Eng Chong 1,4 *, Foo Gen Teck 3,5 , Wong Sher Ming 3,5 , Noorsaadah Abd Rahman 2,4 , Norzulaani Khalid 3,5 , Saiful Anuar Karsani 3,4 , Shatrah Othman 1,4 and Rohana Yusof 1,4 1 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2 Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 3 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 4 Drug Design and Development Research Group, University of Malaya, 50603 Kuala Lumpur, Malaysia. 5 Biotechnology and Bioproduct Research Cluster (UMBIO), University of Malaya, 50603 Kuala Lumpur, Malaysia. Accepted 19 May 2011 Boesenbergia rotunda belongs to the Zingiberaceae family and is abundant in the Southeast Asia. It is widely used as food ingredient and traditional medicine. Biologically, the plant extract contains pharmaceutically important bioactive compounds that exhibit anti-HIV protease, antibacterial, antifungal, anti-inflammatory, anti-tumor and antioxidant activities. Proteomics approaches to study the proteins and/or enzymes involved in the biosynthesis of these compounds are challenging due to the complexity of plant samples and the presence of interfering substances. Here, we describe the development a highly robust and reproducible two-dimensional gel electrophoresis (2DE) protocols for resolving the proteome of B. rotunda suspension cultures. Key words: Two dimensional electrophoresis, plant proteomics, Boesenbergia rotunda, Temu kunci, callus. INTRODUCTION Boesenbergia rotunda (formerly known as Kaempferia pandurata Roxb. or Boesenbergia pandurata (Roxb. Schltr.) is a small perennial monocotyledonous plant belonging to the Zingiberaceae family. It is traditionally known as Chinese keys, Temu Kunci or Fingerroot ginger. B. rotunda is well-known as folk medicine and food ingredient (either as a spice or condiment) in South East Asia. Tubers of B. rotunda are used as a traditional remedy for tumours, swellings, wounds as well as for treating colic disorder such as diarrhea and worms, removing blood clots and as an aphrodisiac. A number of bioactive compounds have been identified from the rhizome extract of B. rotunda. These include boesenbergin, cardamonin, pinostrobin, pinocembrin, panduratin A and 4-hydroxypanduratin A (Jaipetch et al., 1982) that exhibit antioxidant, antibacterial, antifungal, *Corresponding author. E-mail: techong@siswa.um.edu.my, rohana@um.edu.my or saiful72@um.edu.my. Tel: (+603- 79674716). Fax: +603-79674957. anti-inflammatory, antitumor and anti-tuberculosis activities. The antiviral activity of Panduratin A and 4- hydroxypanduratin A in B. rotunda has been demonstrated (Kiat et al., 2006). These compounds have been shown to possess significant inhibitory activities against NS2B/NS3 Dengue viral protease which is required for viral replication. However, their presence in B. rotunda is scarce and the synthesis is likely to involve many proteins and complex pathways. Plant tissue culture and metabolite engineering offer means for exploiting important metabolites and their biosynthesis so that they can be produced in a sustainable fashion. An understanding of the biosynthesis pathways involved can be explored using proteomics. Plants at different developmental stage exhibit temporal and spatial regulation of protein expression (Barbier-Brygoo and Joyard 2004; Hennig, 2007; Klein and Thongboonkerd, 2004; Qureshi et al., 2007). The expression of proteins changes when cells are exposed to different environments (Zang and Komatsu, 2007). There are limited numbers of studies on plant proteomics (rice, Arabidopsis, barley and etc.) (Ferreira et al., 2006;