Abstract
BackgroundMarine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method.MethodsThree Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches.ResultsAny of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins.ConclusionsTrizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile.
Highlights
Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors
Protein extraction Five extraction methods were selected in the present study, including Trizol method, lysis buffer method, Trichloroacetic acid (TCA)/acetone precipitation, Aliquot Trizol method and Trizol method with a commercial clean-up kit (Fig. 1)
The number of spots on the Two-dimensional gel electrophoresis (2-DE) protein profiles from Trizol method with a commercial clean-up kit was almost 13% higher than that from Trizol method alone and was comparable to the largest spot number, which was achieved using lysis buffer method (Fig. 3)
Summary
Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. This is useful for the proteomic study of non-model organisms [8]. 2-DE remains a useful tool for comparative protein profiling [9,10,11,12]
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