Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis

Fic, Ewelina; Kędracka–Krok, Sylwia; Jankowska, Urszula; Piróg, Artur; Dziedzicka-Wasylewska, Marta

doi:10.1002/elps.201000197

Cited by 173 publications

(114 citation statements)

References 16 publications

(17 reference statements)

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“…It generally delivers high-protein recovery as described by Fic et al (2010) when comparing various precipitation techniques for brain tissue. The protein solution to be precipitated must be perfectly clear.…”

Section: Precipitation With Chloroform/methanolmentioning

confidence: 98%

Detailed Methodologies and Protocols

Righetti¹,

Boschetti

2013

Low-Abundance Proteome Discovery

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Section: Precipitation With Chloroform/methanolmentioning

confidence: 98%

Detailed Methodologies and Protocols

Righetti¹,

Boschetti

2013

Low-Abundance Proteome Discovery

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“…Proteins (80 mg) from the supernatant were precipitated by the methanol/ chloroform method (Fic et al, 2010), after which they were denatured in 7 M urea/2 M thiourea/100 mM TEAB (pH 7.5) and reduced with 50 mM Tris(2-carboxyethyl) phosphine (pH 8) for 60 min at 37°C. Cys residues were then alkylated with 200 mM methyl methanethiosulfonate (Pierce MMTS; Thermo Fisher Scientific) for 10 min at room temperature.…”

Section: Enzymatic Digestion and Tmt-6 Plex Labelingmentioning

confidence: 99%

Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms

et al. 2016

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Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the CalvinBenson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

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“…For insoluble protein extraction and analysis, Acetone/TCA precipitation protocol was used (Fic et al, 2010). Briefly, insoluble proteins were homogenizing with 70% formic acid (Thermo Fisher Scientific, Rockford, IL), followed by centrifugation at 100,000 ×g for 1 h. Supernatant was collected and the formic acid of the supernatant was neutralized using the following ration (1 volumen of sample, 1 volumen of neutralization buffer and 1.8 volumenes 10 N of NaOH).…”

Section: Tissue Preparationmentioning

confidence: 99%

Endogenous murine tau promotes neurofibrillary tangles in 3xTg-AD mice without affecting cognition

Baglietto‐Vargas

Kitazawa

et al. 2014

Neurobiology of Disease

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Recent studies on tauopathy animal models suggest that the concomitant expression of the endogenous murine tau delays the pathological accumulation of human tau, and interferes with the disease progression. To elucidate the role of endogenous murine tau in a model with both plaques and tangles, we developed a novel transgenic mouse model by crossing 3xTg-AD with mtauKO mice (referred to as 3xTg-AD/mtauKO mice). Therefore, this new model allows us to determine the pathological consequences of the murine tau. Here, we show that 3xTg-AD/mtauKO mice have lower tau loads in both soluble and insoluble fractions, and lower tau hyperphosphorylation level in the soluble fraction relative to 3xTg-AD mice. In the 3xTg-AD model endogenous mouse tau is hyperphosphorylated and significantly co-aggregates with human tau. Despite the deletion of the endogenous tau gene in 3xTg-AD/mtauKO mice, cognitive dysfunction was equivalent to 3xTg-AD mice, as there was no additional impairment on a spatial memory task, and thus despite increased tau phosphorylation, accumulation and NFTs in 3xTg-AD mice no further effects on cognition are seen. These findings provide better understanding about the role of endogenous tau to Alzheimer's disease (AD) pathology and for developing new AD models.

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Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis

Cited by 173 publications

References 16 publications

Detailed Methodologies and Protocols

Detailed Methodologies and Protocols

Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms

Endogenous murine tau promotes neurofibrillary tangles in 3xTg-AD mice without affecting cognition

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