Production of high-quality two-dimensional gel electrophoresis profile for marine medaka samples by using Trizol-based protein extraction approaches

Abstract

Background: 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. 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. Methods: Three 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. Results: Any 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. Conclusions: Trizol-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.

High-yield Skeletal Muscle Protein Recovery from TRIzol After RNA and DNA Extraction

“…We confirmed that the currently presented isolation protocol is compatible with subsequent western blot analysis of total and phosphorylated proteins, which is consistent with previous reports showing that protein from TRIzol samples retains good quality for downstream analyses [ 6 , 26–28 ]. Our method can help laboratories routinely study changes in protein species from TRIzol samples that would otherwise be thrown away.…”

A Device for Stereotaxic Viral Delivery into the Brains of Neonatal Mice

“…We confirmed that the currently presented isolation protocol is compatible with subsequent western blot analysis of total and phosphorylated proteins, which is consistent with previous reports showing that protein from TRIzol samples retains good quality for downstream analyses [6,[26][27][28]. Our method can help laboratories routinely study changes in protein species from TRIzol samples that would otherwise be thrown away.…”

“…We obtained complete dissolution of the protein pellet without special treatment or equipment and achieved higher yield relative to direct homogenization using RIPA lysis buffer. Despite significant interest in simultaneous isolation of RNA, DNA and protein, most other previous methods only recover a small fraction of the precipitated proteins [1,[5][6][7]26]. The higher yield even compared with direct homogenization in RIPA buffer may be explained by improved solubility of large sarcomeric proteins (e.g., myosin, nebulin and titin) while still recovering all of the smaller protein species.…”

Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi

“…The generation of high-quality 2-DE profiles with well-resolved proteins relies on a suitable protein extraction method, which is usually species-specific. Several extraction methods were previously tested on medaka by our team, and TRIzol extraction followed by a commercial 2D clean-up kit showed the best overall results with the highest resolution of protein spots and the lowest background signal [78]. Protein extraction with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) followed the user manual provided by the manufacturer, with a small modification suggested in a previous study [79].…”