LeHavreV1, Main, Exploration, bibRecord, 000473

Comparison of Protein-Extraction Methods for Gills of the Shore Crab, Carcinus maenas (L.), and Application to 2DE

Identifieur interne : 000473 ( Main/Exploration ); précédent : 000472; suivant : 000474

Comparison of Protein-Extraction Methods for Gills of the Shore Crab, Carcinus maenas (L.), and Application to 2DE

Auteurs : François Panchout ; Julie Letendre ; Florence Bultelle ; Xavier Denier ; Béatrice Rocher ; Philippe Chan ; David Vaudry [France] ; Fabrice Durand [France]

Source :

Journal of Biomolecular Techniques : JBT [ 1524-0215 ] ; 2013.

RBID : PMC:3792702

Abstract

As it is well-established that protein extraction constitutes a crucial step for two-dimensional electrophoresis (2DE), this work was done as a prerequisite to further the study of alterations in the proteome in gills of the shore crab Carcinus maenas under contrasted environmental conditions. Because of the presence of a chitin layer, shore crab gills have an unusual structure. Consequently, they are considered as a hard tissue and represent a challenge for optimal protein extraction. In this study, we compared three published extraction procedures for subsequent applications to 2DE: the first one uses homogenization process, the second one included an additional TCA-acetone precipitation step, and finally, the third one associated grinding in liquid nitrogen (N₂) and TCA-acetone precipitation. Extracted proteins were then resolved using 1DE and 2DE. Although interesting patterns were obtained using 1DE with the three methods, only the one involving grinding in liquid N₂ and TCA-acetone precipitation led to proper resolution after 2DE, showing a good level of reproducibility at technical (85%) and biological (84%) levels. This last method is therefore proposed for analysis of gill proteomes in the shore crab.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792702

DOI: 10.7171/jbt.13-2404-002
PubMed: 24294114
PubMed Central: 3792702

Affiliations:

Le document en format XML

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<front><div type="abstract" xml:lang="en"><p>As it is well-established that protein extraction constitutes a crucial step for two-dimensional electrophoresis (2DE), this work was done as a prerequisite to further the study of alterations in the proteome in gills of the shore crab <italic>Carcinus maenas</italic>
 under contrasted environmental conditions. Because of the presence of a chitin layer, shore crab gills have an unusual structure. Consequently, they are considered as a hard tissue and represent a challenge for optimal protein extraction. In this study, we compared three published extraction procedures for subsequent applications to 2DE: the first one uses homogenization process, the second one included an additional TCA-acetone precipitation step, and finally, the third one associated grinding in liquid nitrogen (N<sub>2</sub>
) and TCA-acetone precipitation. Extracted proteins were then resolved using 1DE and 2DE. Although interesting patterns were obtained using 1DE with the three methods, only the one involving grinding in liquid N<sub>2</sub>
 and TCA-acetone precipitation led to proper resolution after 2DE, showing a good level of reproducibility at technical (85%) and biological (84%) levels. This last method is therefore proposed for analysis of gill proteomes in the shore crab.</p>
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Comparison of Protein-Extraction Methods for Gills of the Shore Crab, Carcinus maenas (L.), and Application to 2DE

Comparison of Protein-Extraction Methods for Gills of the Shore Crab, Carcinus maenas (L.), and Application to 2DE

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