Evaluating the compatibility of three colorimetric protein assays for two‐dimensional electrophoresis experiments

Kao, Shao‐Hsuan; Wong, H. Y.; Chiang, C.; Chen, Han-Min

doi:10.1002/pmic.200700600

Cited by 24 publications

(17 citation statements)

References 32 publications

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“…Fresh elution buffer was added to the bead and the above process was repeated twice to ensure maximum protein recovery. The released proteins were pooled and the resulting enriched protein samples were analyzed for protein content using the bicinchoninic acid method (Kao et al, 2008).…”

Section: Enrichment Of Minor Abundance Proteinsmentioning

confidence: 99%

Comparative proteomics analysis of plasma proteins during the transition period in dairy cows with or without subclinical mastitis after calving

Yang¹,

Wang²,

Bu³

et al. 2012

CZECH J ANIM SCI

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ABSTRACT:The transition period is the most critical time of the cow's lactation cycle that is associated with the onset of mastitis. In this study, changes of plasma proteins in cows (n = 12) with or without subclinical mastitis after calving were determined by two-dimensional electrophoresis (2-DE), which detected 18 spots with variations in protein spots abundance. These spots were identified by liquid chromatography coupled with tandem mass spectrometry. The changes in protein profile from day 21 before calving to day 1 after calving were similar in cows with or without subclinical mastitis. Abundance of α1 acid glycoprotein (AGP) and haptoglobin was dramatically increased at parturition, while transthyretin was down-regulated at parturition, and apolipoprotein E and immunoglobulin gamma 1 were up-regulated at postpartum compared with prepartum in periparturient dairy cows. In cows infected with subclinical mastitis, AGP, haptoglobin, and serum amyloid A were dramatically increased and continued to be elevated in plasma from day 1 to day 21 after calving compared with cows free of mastitis. Changes of protein in plasma at parturition may serve as an immune system response to parturition and lactation process at the protein level and suggest that these altered proteins would not serve as a potential marker for predicting if the periparturient dairy cows are susceptible to subclinical mastitis.

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Section: Enrichment Of Minor Abundance Proteinsmentioning

confidence: 99%

Comparative proteomics analysis of plasma proteins during the transition period in dairy cows with or without subclinical mastitis after calving

Yang¹,

Wang²,

Bu³

et al. 2012

CZECH J ANIM SCI

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show abstract

“…50 mg white powder was resuspended in 800 l SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, 1 mM PMSF, pH7.6, including one-fold PhosSTOP phosphatase inhibitor mixture from Roche), and boiled for 15 min in water bath, and followed by 100 s of sonication. After centrifugation at 14,000 ϫ g for 15 min at 4°C, the protein in supernatant was quantified via BCA (bicinchoninic acid) method (30).…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars

Hu³

et al. 2016

Molecular & Cellular Proteomics

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Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants.In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at threetrifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mM NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier

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“…Proteins precipitated by TCA-actone were dissolved in a buffer containing 6 M urea, 2 M thiourea, 0.02 M DTT, 2% CHAPS, 0.2% Bio-lyte (pH 3-10), and 0.05% IPG buffer, and protein concentrations were determined using the Bradford method (Kao et al 2008).…”

Section: Methodsmentioning

confidence: 99%

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of Dunaliella salina

Cui

Chai

et al. 2009

J Appl Phycol

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The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P<0.01) by 2.5 M and 14-fold higher (P<0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.

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Evaluating the compatibility of three colorimetric protein assays for two‐dimensional electrophoresis experiments

Cited by 24 publications

References 32 publications

Comparative proteomics analysis of plasma proteins during the transition period in dairy cows with or without subclinical mastitis after calving

Comparative proteomics analysis of plasma proteins during the transition period in dairy cows with or without subclinical mastitis after calving

Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of Dunaliella salina

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