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Type: Artigo de periódico
Title: Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease
Author: Pirovani, CP
Carvalho, HAS
Machado, RCR
Gomes, DS
Alvim, FC
Pomella, AWV
Gramacho, KP
Cascardo, JCD
Pereira, GAG
Micheli, F
Abstract: Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. in order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction - under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF - using infiltration-centrifugation procedure - that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.
Subject: apoplastic washing fluid
cacao vegetative organs
enzymatic activity
plant protein extraction
two-dimensional gel electrophoresis
Country: Alemanha
Editor: Wiley-v C H Verlag Gmbh
Citation: Electrophoresis. Wiley-v C H Verlag Gmbh, v. 29, n. 11, n. 2391, n. 2401, 2008.
Rights: fechado
Identifier DOI: 10.1002/elps.200700743
Date Issue: 2008
Appears in Collections:Unicamp - Artigos e Outros Documentos

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