A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis : A follow up
With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan. © 2006 Elsevier B.V. All rights reserved.
| Main Authors: | , , , , , |
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| Format: | article biblioteca |
| Language: | eng |
| Subjects: | Q04 - Composition des produits alimentaires, Citrus, protéine, extraction, séparation, électrophorèse, pH, http://aims.fao.org/aos/agrovoc/c_1637, http://aims.fao.org/aos/agrovoc/c_6259, http://aims.fao.org/aos/agrovoc/c_36910, http://aims.fao.org/aos/agrovoc/c_6973, http://aims.fao.org/aos/agrovoc/c_2523, http://aims.fao.org/aos/agrovoc/c_5753, |
| Online Access: | http://agritrop.cirad.fr/539069/ http://agritrop.cirad.fr/539069/1/document_539069.pdf |
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| Summary: | With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan. © 2006 Elsevier B.V. All rights reserved. |
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