Журнал протеомики и биоинформатики
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ISSN: 0974-276X
ISSN: 0974-276X
Muhammad Tahir and Samina Noor Shakeel
Two-dimensional (2D) gel electrophoresis is a powerful technique used for the differential protein expression analysis. High-resolution two-dimensional electrophoresis is still the main applied separation technique in proteomics. A successful 2D gel electrophoresis depends on the proper optimized conditions most importantly an optimized buffer. In this study we checked different factors affecting 2D gel results including rehydration time, reducing agents, chaotrophic agents, and detergents. We chose Cynodon dactylon as an experimental plant. Proteins were extracted and purified. For the proper solubilization of the protein sample different rehydration buffers of pH 3-10 were applied having different amount of chaotrophic agents like urea and thiourea, detergents (NP-40, Triton X-100 and CHAPS) and reducing agents (β-merceptoethanol and DTT). To further improve the protein profile on 2D gel, solubilized proteins were re-extracted, washed and re-solubilized in a fresh rehydration buffer. Our modified protocol showed a high increase in protein solubility and improvement in protein 2D gel profile for C. dactylon proteome. In conclusion, we have successfully optimized the conditions for C. dactylon proteome analysis by 2D gel electrophoresis. These optimized conditions will lay the basis for further profound studies in the field of proteomics.