A novel method of protein extraction from perennial <b><i>Bupleurum</i></b> root for 2‐DE

Xie, Hui; Pan, Shengli; Liu, Shufen; Ye, Kan; Huo, Keke

doi:10.1002/elps.200600354

Cited by 23 publications

(20 citation statements)

References 17 publications

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“…2-DE is the most widely used proteomic platform, but plant proteins are typically more difficult to resolve by 2-DE than those from other organisms, because of the abundance of interfering compounds, in particular secondary metabolites [21]. These reduce electrophoretic resolution by creating horizontal or vertical streaking and smearing, thereby restricting the number of protein spots that can be detected [22]. To overcome this problem, DiVaPRA, Plant Genetics and Breeding -University of Torino, Grugliasco, Italy extraction methods are required, which eliminate as many of the interfering compounds as possible.…”

Section: Introductionmentioning

confidence: 99%

Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

et al. 2009

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Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana. Keywords IntroductionThe globe artichoke (Cynara cardunculus L. var. scolymus) is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Globe artichoke is mainly used in human nutrition: its immature inflorescence (capitulum, or more commonly ''head'' or ''bud'') represents the edible part of the plant and is consumed fresh, canned or frozen. It is also a promising source of biopharmaceuticals [1], such as luteolin and mono-/ di-caffeoylquinic acids [2,3]. These compounds are responsible for its therapeutic effects, which include hepatoprotective and choleretic activity [4], protection against oxidative damage caused by free radicals [5], inhibition of cholesterol biosynthesis and prevention of arteriosclerosis and other vascular disorders [6,7], inhibition of HIV integrase [8,9] and antibacterial and antifungal activity [10]. Because of the current interest in nutraceuticals for human healthcare, there has been a growing focus on the identity of the genes and networks involved in secondary metabolism. Only a limited number of genes involved in phenylpropanoid and sesquiterpene lactone metabolism has been isolated so far [11,12]. Recently, a set of basic proteomic tools has been applied to purify a soluble peroxidase (artichoke leaves soluble peroxidase) from globe artichoke leaves [13], but its proteome as a whole has not been explored. Although molecular markers have been developed [14][15][16][17] and applied to produce a genetic map [18], the globe artichoke genome also remains poorly researched.The genome is fixed in time, but the proteome is very plastic, depending on tissue type, developmental stage and age, and is also strongly modulated by the environment. The differential response of related proteomes to the same set of biotic and abiotic factors allows the genetic mapping of expressed genes [19]. These comparisons can also identify ''candidate proteins'' involved in the determination of a particular phenotyp...

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Section: Introductionmentioning

confidence: 99%

Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

et al. 2009

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“…Roots, especially of recalcitrant species such as T. cacao (Figueira et al, 1994;Gesteira et al, 2003), besides being an organ containing tissues that are difficult of macerate, have low protein content (Isaacson et al, 2006;Xie et al, 2007). Roots have many non-protein contaminants that affect 2-DE, including polysaccharides, polyphenols, nucleic acids, terpenes, and organic acids; these contaminants accumulate mainly in the vacuole in various soluble forms (Tsugita and Kamo, 1999;Pan, 2000).…”

Section: Discussionmentioning

confidence: 99%

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses

Bertolde¹,

Almeida²,

Silva³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Efficient method of protein extraction from cocoa rootsProteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

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“…De acordo com relato de Caetano et al (2007), o tampão sacarose (De Mot & Vanderleyden, 1989) também se mostrou eficiente na extração de proteínas de raízes de feijão-corda infectadas com Meloidogyne incognita, visto que amostra obtida a partir deste tampão, avaliada por eletroforese bi-dimensional, apresentou 250 proteínas, em comparação com a amostra obtida a partir do tampão relatado por Xie et al (2007) que apresentou 200 proteínas. Os dados gerados no presente trabalho não nos permitem estimar os números de proteínas obtidas com os diferentes tampões, visto que as análises ficaram restritas a géis uni-dimensionais, contudo, Figura 2 -Análise eletroforética (sds-PAGE) de extratos protéicos (3 μg) de raízes de P. tuberculatum Jacq.…”

Section: Folhas Em Tampão Salino 896unclassified

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

Silva

Souza

2009

Acta Amaz.

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O Estado do Pará é o principal produtor brasileiro de pimenta-do-reino (Piper nigrum Link), entretanto a sua produção tem sido bastante afetada pela doença conhecida como fusariose. O Fusarium solani f. sp. piperis é o agente causador desta doença que afeta o sistema radicular da planta, causando o apodrecimento das raízes e a queda das folhas levando à morte da planta. Algumas piperáceas nativas da região amazônica, entre elas a espécie Piper tuberculatum Jacq., têm se mostrado resistentes à infecção pelo F. solani f. sp. piperis, e desta forma têm sido utilizadas em estudos de interação planta-patógeno. Neste trabalho foram avaliadas cinco condições de extração de proteínas com o objetivo de selecionar tampões adequados para a extração de proteínas totais de folhas e raízes de P. tuberculatum. Os tampões utilizados para a extração de proteínas de raízes e folhas foram: tampão salino, tampão sacarose, tampão glicerol, tampão uréia e tampão fosfato de sódio. As análises quantitativas mostraram que os tampões sacarose, glicerol e uréia foram mais eficientes na extração de proteínas de folhas e raízes. Análises de SDS-PAGE mostraram padrões diferenciados de bandas em extratos protéicos de folhas e raízes obtidos com os diferentes tampões. Os resultados obtidos neste trabalho contribuem para a identificação de tampões de extração adequados para a obtenção de amostras de proteínas totais em estudos de interação P. tuberculatum -F. solani f. sp. piperis. Extraction and polyacrylamide gels electrophoresis analysis (SDS-PAGE) of total protein from leaves and roots of Piper tuberculatum ABSTRACTThe Pará State is the principal Brazilian producer of black pepper (Piper nigrum Link), however, the spice's production has been damaged by the Fusarium disease. The Fusarium solani f. sp. piperis, causal agent of this disease which attacks the plant root system promoting the root rot, leaves fall and plant death. Piperaceae plants endemic of Amazon region and resistant to F. solani f. sp. piperis infection, such as Piper tuberculatum Jacq., which has been studied in order to understand this host-pathogen interaction. In this work were evaluated five conditions for total protein extraction aiming to select suitable buffers for extraction of total proteins from P. tuberculatum leaves and roots. The conditions used to roots and leaves protein extractions were salt buffer, sucrose buffer, glycerol buffer, urea buffer and sodium phosphate buffer. Quantitative analysis showed that sucrose, glycerol and urea buffers were more efficient for leaves and roots protein extractions. SDS-PAGE analysis showed distinct band patterns in leaves and roots protein extracts obtained with different buffers. Our results could support the selection of extraction buffers for proteomics analysis in P. tuberculatum -F. solani f sp. piperis interaction studies.

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A novel method of protein extraction from perennial Bupleurum root for 2‐DE

Cited by 23 publications

References 17 publications

Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

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