Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Cho, Kyoungwon; Torres, Nilka Lineth; Subramanyam, Subhashree; Deepak, S. A.; Sardesai, Nagesh; Han, Oksoo; Williams, Christie E.; Ishii, Hideo; Iwahashi, Hitoshi; Rakwal, Randeep

doi:10.1007/bf03031120

Cited by 34 publications

(29 citation statements)

References 24 publications

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“…Briefly, proteins were precipitated for 1 h at -207C in four volumes of ice-cold acetone (2207C), followed by centrifugation at 15 000 rpm for 15 min at 47C. The supernatant was decanted, and the pellet was washed twice with wash buffer (acetone containing 0.07% 2-ME, 2 mM EDTA, and two EDTA-free proteinase inhibitor cocktail tablets in a final volume of 100 mL buffer), followed by removal of all the acetone by air drying the pellet at ambient RT; the pellet was kept at -807C for at least 24 h. The pellet was subsequently solubilized in lysis buffer (LB) containing thiourea and Tris (LB-TT: 7 M urea, 2 M thiourea, 4% w/v 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 18 mM TrisHCl (pH 8.0), 14 mM Trizma base, two EDTA-free proteinase inhibitor cocktail tablets, 0.2% v/v Triton X-100 (R), 50 mM DTT to a final volume of 100 mL [35]) and incubated for 20 min at 47C with occasional vortexing and sonication, and centrifuged at 5000 rpm for 15 min at 107C. The supernatant was used for protein determination using a Coomassie Plus ™ protein assay kit, and stored in aliquots at -807C until analyzed by 2-DE.…”

Section: -Dementioning

confidence: 99%

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Gel‐based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

et al. 2007

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We examined responses of cultivated bean (Phaseolus vulgaris L. cv. IDIAP R-3) and maize (Zea mays L. cv. Guarare 8128) plants exposed to ozone (O(3)) using a leaf injury assessment and proteomics approach. Plants grown for 16 days in greenhouse were transferred to an O(3) chamber and exposed continuously to 0.2 ppm O(3) or filtered pollutant-free air for up to 72 h. CBB-stained gels revealed changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein. By Western analysis changes in marker proteins for O(3) damage in leaves by 1-DE were checked. In bean leaves, two superoxide dismutase (SOD) protein (19 and 20 kDa) were dramatically decreased, while ascorbate peroxidase (APX, 25 kDa), small heat shock protein (HSP, 33 kDa), and a naringenin-7-O-methyltransferase (NOMT, 42 kDa) were increased by O(3). In maize leaves, expression levels of catalase (increased), SOD (decreased), and APX (increased) were drastically changed by O(3) depending on the leaf stage, whereas crossreacting HSPs (24 and 30 kDa) and NOMT (41 kDa) proteins were strongly increased in O(3)-stressed younger leaves. These results indicated a clear modulation of oxidative stress-, heat shock-, and secondary metabolism-related proteins by O(3). Finally, 2-DE at 72 h after O(3) exposure revealed changes (induction/suppression) in expression levels of 25 and 12 protein spots in bean and maize leaves, respectively. Out of these, ten and nine nonredundant proteins in bean and maize, respectively, were identified by MS. A novel pathogenesis-related protein 2 may serve as a potential marker for O(3) stress in bean.

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Section: -Dementioning

confidence: 99%

“…The homogenates were centrifuged at 18,5006g once for 5 min and then for 10 min at 47C. The supernatant was used for protein determination using a Coomassie Plus ™ (Pierce, Rockford, IL, USA) protein assay kit [35]. Samples for SDS-PAGE (also referred to as 1-DE) were prepared as described previously [34].…”

Section: -De and Western Analysismentioning

confidence: 99%

Gel‐based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

et al. 2007

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“…Effective protein extraction, solubilisation, and purification from plant tissues are essential for a good and reproducible 2-DE approach, as the amount and quality of extracted proteins ultimately determine the protein spot number, resolution and intensity. Important characteristics of a good protein isolation method are the reproducible capture and solubilisation of a full set of proteins from a certain sample with minimal post-extraction artefact formation, proteolytic degradation and contamination with non-protein molecules (72,73). In plant proteomics, there are several factors that can affect the method chosen for protein extraction, such as plant species, tissue type, organ, cell, organelle, and the chemical and physical properties of the observed proteins.…”

Section: Cvjetko Et Al Proteomics Of Heavy Metal Toxicity In Plantsmentioning

confidence: 99%

Proteomics of heavy metal toxicity in plants

Cvjetko

Zovko²,

Balen

2014

Archives of Industrial Hygiene and Toxicology

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Plants endure a variety of abiotic and biotic stresses, all of which cause major limitations to production. Among abiotic stressors, heavy metal contamination represents a global environmental problem endangering humans, animals, and plants. Exposure to heavy metals has been documented to induce changes in the expression of plant proteins. Proteins are macromolecules directly responsible for most biological processes in a living cell, while protein function is directly influenced by posttranslational modifications, which cannot be identified through genome studies. Therefore, it is necessary to conduct proteomic studies, which enable the elucidation of the presence and role of proteins under specific environmental conditions. This review attempts to present current knowledge on proteomic techniques developed with an aim to detect the response of plant to heavy metal stress. Significant contributions to a better understanding of the complex mechanisms of plant acclimation to metal stress are also discussed.

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“…The ideal extraction method should reproducibly capture the most comprehensive repertoire of proteins possible while minimizing protein degradation and nonprotein contamination [7]. Over the past 30 years, with the constant improvement of 2DE technology, many sample preparation methods for plant tissues have been developed for enhanced 2DE and proteomic analysis [e.g., 1,[8][9][10][11][12][13][14][15][16][17]. These methods are typically based on trichloroacetic acid (TCA)/acetone precipitation and phenol extraction.…”

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Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Gong

Wang

2014

Proteomics

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Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE-based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).

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Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Cited by 34 publications

References 24 publications

Gel‐based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

Gel‐based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

Proteomics of heavy metal toxicity in plants

Protein extraction from plant tissues for 2DE and its application in proteomic analysis

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