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Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

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Abstract

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.

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Abbreviations

DTT:

Dithiothreitol

PR-10:

Pathogenesis-related class 10

PVDF:

Polyvinylidene difluoride

PVPP:

Polyvinylpolypyrrolidone

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TCA:

Trichloroacetic acid

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Acknowledgments

The authors would like to thank Prof. Dr. Eva Čellárová and Dr. Ján Košuth (Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Košice, Slovakia) for kindly providing the in vitro plants of different Hypericum species. This research was financially supported by grants from Finnish Cultural Foundation and Niemi Foundation.

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Authors and Affiliations

  1. Department of Biology, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland

    Katja Karppinen, Erja Taulavuori & Anja Hohtola

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  1. Katja Karppinen
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  2. Erja Taulavuori
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  3. Anja Hohtola
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Correspondence to Katja Karppinen.

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Karppinen, K., Taulavuori, E. & Hohtola, A. Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development. Mol Biotechnol 46, 219–226 (2010). https://doi.org/10.1007/s12033-010-9299-9

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  • DOI: https://doi.org/10.1007/s12033-010-9299-9

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