Improved two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients

Davidsen, Nina B.

doi:10.1002/elps.1150170718

Cited by 4 publications

(1 citation statement)

References 5 publications

(1 reference statement)

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“…Maybe more proteins should be affected by stresses. One possible reason is that in the plant leaf sample, RuBisCO is the most abundant protein (about 50% of soluble protein) that has hindered high resolution of 2-DE due to the limitations of the loading capacity on the IEF gel (des Francs et al 1985), and low abundance proteins present or induced in leaf samples remain difficult to analyze, despite the sensitivity of current instrumentation and high loading capacity of immobilized pH gradient strips (IPGs) (Davidsen 1996). Therefore, it is necessary to enrich these low abundance proteins such as membrane proteins prior to analysis.…”

Section: Discussionmentioning

confidence: 99%

Proteins responding to drought and high-temperature stress in Populus × euramericana cv. ‘74/76’

Jian-guo

Duan

et al. 2008

Trees

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Proteomic analysis provides a powerful method of studying plant responses to stress at the protein level. In order to study stress-responsive molecular mechanisms for Populus 9 euramericana cv. '74/76', one of the most important forest plantation tree species in subtropical and temperate regions, we analyzed the response of 2-year-old cuttings of P. 9 euramericana cv. '74/76' to drought and high temperature using twodimensional gel electrophoresis. More than 1,000 reproducible leaf proteins were detected in the controls and treatments, and 26 proteins were found to change notably in abundance. We identified 13 proteins affected by drought stress and 11 proteins affected by high temperature. These proteins are mainly involved in photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and putative photosystem I reaction center subunit II precursor, and detoxification (manganese superoxide dismutase and methionine sulfoxide reductase A). Furthermore, the level of the photosynthesis proteins affected greatly by the imposed stress conditions was consistent with the observed noticeable decrease in net photosynthesis rate. These studies provides a fundamental data for future research on responses to drought and high temperature, two major factors limiting the growth of forest trees during summer under recent climatic warming.

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Section: Discussionmentioning

confidence: 99%

Proteins responding to drought and high-temperature stress in Populus × euramericana cv. ‘74/76’

Jian-guo

Duan

et al. 2008

Trees

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Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays

et al. 2001

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Two-dimensional electrophoresis (2-DE) is known as the most effective as well as one of the simplest methods for separating proteins. However, a few hundred plant leaf proteins out of thousands visualized on a 2-DE gel can be identified by chemical analysis due to the presence of ribulose bisphosphate carboxylase/oxygenase (Rubisco) that limits protein loading. We describe the extraction and fractionation technique with polyethylene glycol (PEG) to analyze rice leaf proteins. Rice proteins were extracted with Mg/NP-40 extraction buffer. The Mg/Nonidet P-40 (NP-40) buffer extract was further fractionated with PEG into three fractions: 10% PEG and 10-20% PEG precipitants and the final supernatant fraction that was precipitated with acetone. Rubisco, the most abundant rice leaf protein, was enriched in the 20% PEG precipitant. This fractionation technique analyzed at least 2,600 well-separated protein spots and exhibited less than 1.2% of noticeable overlapping spots. An immunological approach was used to verify the efficiency whether PEG fractionation technique can detect or enrich signal transduction components such as Galpha, ADP ribosylation factor, small GTP binding protein and 14-3-3. The ADP ribosylation factor (ARF) and Galpha were only detected in the PEG supernatant fraction not in the total protein fraction. The small GTP binding protein (Rab 7) was identified in the 10% PEG fraction and only faintly in the total protein fraction. The 14-3-3 protein was detected in all fractions but was especially prevalent in the 20% PEG fraction.

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2D PAGE: Sample Preparation and Fractionation

Posch¹

2008

Methods in Molecular Biology™

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Improved two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients

Cited by 4 publications

References 5 publications

Proteins responding to drought and high-temperature stress in Populus × euramericana cv. ‘74/76’

Proteins responding to drought and high-temperature stress in Populus × euramericana cv. ‘74/76’

Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays

2D PAGE: Sample Preparation and Fractionation

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