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Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis

Nature Protocols volume 9, pages 362–374 (2014)Cite this article

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Abstract

Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis. Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.

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Figure 1: Schematic overview of the full protocol.
Figure 2: Quality control and 2-DE separation of maize leaf and embryo proteins.
Figure 3: Comparative 2-DE analysis of maize leaves proteins by using three extraction protocols.

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Acknowledgements

Work in our laboratory was supported by grants from the National Natural Science Foundation of China (grant nos. 31230055 and 31371543) and the Ministry of Science and Technology of China (grant no. 2011CB111509).

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Authors and Affiliations

  1. Synergetic Innovation Center of Henan Grain Crops, Key Laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, College of Life Sciences, Henan Agricultural University, Zhengzhou, China

    Xiaolin Wu, Erhui Xiong & Wei Wang

  2. Department of Life Sciences, University of Siena, Siena, Italy

    Monica Scali & Mauro Cresti

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  1. Xiaolin Wu
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  2. Erhui Xiong
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  3. Wei Wang
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Contributions

X.W. and E.X. conducted the experiments and interpreted the data. X.W. and W.W. developed the protocol, interpreted the data and drafted the manuscript. M.S. and M.C. developed the sections of the protocol and provided intellectual and technical expertise.

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Correspondence to Wei Wang.

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Wu, X., Xiong, E., Wang, W. et al. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat Protoc 9, 362–374 (2014). https://doi.org/10.1038/nprot.2014.022

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