scholarly journals Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots

2015 ◽  
Vol 87 (3) ◽  
pp. 1853-1863 ◽  
Author(s):  
MILCA B. VILHENA ◽  
MÔNICA R. FRANCO ◽  
DAIANA SCHMIDT ◽  
GISELLE CARVALHO ◽  
RICARDO A. AZEVEDO
Keyword(s):  
Gel Electrophoresis ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Protein Yield ◽  
Crucial Aspect ◽  
Large Protein ◽  
Total Proteins ◽  
Solanum Lycopersicum L ◽  
Wide Range ◽  
Leaves And Roots

Proteomics is an outstanding area in science whose increasing application has advanced to distinct purposes. A crucial aspect to achieve a good proteome resolution is the establishment of a methodology that results in the best quality and wide range representation of total proteins. Another important aspect is that in many studies, limited amounts of tissue and total protein in the tissue to be studied are found, making difficult the analysis. In order to test different parameters, combinations using minimum amount of tissue with 4 protocols for protein extraction from tomato (Solanum lycopersicum L.) leaves and roots were evaluated with special attention to their capacity for removing interferents and achieving suitable resolution in bidimensional gel electrophoresis, as well as satisfactory protein yield. Evaluation of the extraction protocols revealed large protein yield differences obtained for each one. TCA/acetone was shown to be the most efficient protocol, which allowed detection of 211 spots for leaves and 336 for roots using 500 µg of leaf protein and 800 µg of root protein per gel.

scholarly journals Comparison of methods for the extraction of proteins from root and leaf tissue of sugarcane (Saccharum spp.) for proteomic analysis

2020 ◽  
pp. 1221-1229
Author(s):  
Luciana da Silva Viana ◽  
Paulo Pedro da Silva ◽  
Velber Xavier Nascimento ◽  
Alessandro Riffel ◽  
Antônio Euzébio Goulart Sant’Ana
Keyword(s):  
Proteomic Analysis ◽  
Qualitative Data ◽  
Ammonium Acetate ◽  
Control Method ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Cell Wall Polysaccharides ◽  
Total Proteins ◽  
Protein Determination ◽  
Leaves And Roots

The extraction of proteins from plants is a crucial procedure for successful protein determination such as purification, separation, and mass spectrometry. Protein extraction from plant tissues is generally difficult due to the presence of various molecules (cell wall, polysaccharides, and lipids) and interfering compounds. For this reason, the step of separation of proteins is a big challenge in obtaining good results in plant proteomic studies, notably from sugarcane. The current study assesses three extraction methods to prepare protein samples for proteomic analysis. Method 1 (control): TCA/acetone, method 2: TCA/acetone modified and Method 3: Phenol/SDS/ammonium acetate. Plants of cultivar RB92579 were grown in 10L pots under ideal humidity conditions in a greenhouse for 60 days. Samples collected on leaves +1 and roots were carried out using nitrogen and stored in an ultra-freezer at -80ºC for later use in proteome assays. For the tested methods, a comparison was made between the quantitative and qualitative data obtained from the tissue of sugarcane leaves and roots. According to the results obtained, methods 2 and 3 produced the best yield in the extraction of total proteins from the leaves and roots of sugarcane, when compared to (control) method 1 (TCA/acetone). This can be observed when comparing the quantitative and qualitative data obtained using the different extraction methods. By comparing methods 2 and 3, the latter showed a massive gain of extracted proteins much greater than the first method, mainly when the extraction of total proteins from the roots are compared. Similarly, the 2-DE gels run after using method 3 showed less background, compared to method 2. Another observation was the presence of different “spots” in the 2-DE gels between the samples extracted using methods 2 and 3. Method 3 (phenol / SDS / ammonium acetate) presented better results for extraction of proteins and in the 2-DE gels, with a greater number of total and specific “spots”, greater reproducibility and less background. This method could be utilized as the standard method for proteomic studies in sugarcane.

scholarly journals Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1409
Author(s):  
Truong Van Nguyen ◽  
So-Wun Kim ◽  
Cheol-Woo Min ◽  
Ravi Gupta ◽  
Gi-Hyun Lee ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Extraction Methods ◽  
Label Free ◽  
Protein Isolation ◽  
Mva Pathway ◽  
Ginsenoside Biosynthesis ◽  
Wide Range ◽  
Proteomics Approach ◽  
Reproducible Method ◽  
Korean Ginseng

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, phenol–TCA/acetone, and phenol–MeOH/chloroform methods. The TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

Versatile Health Benefits of Catechin from Green Tea (Camellia sinensis)

2019 ◽  
Vol 15 (1) ◽  
pp. 3-10 ◽  
Author(s):  
Satheesh Babu Natarajan ◽  
Suriyakala Perumal Chandran ◽  
Sahar Husain Khan ◽  
Packiyaraj Natarajan ◽  
Karthiyaraj Rengarajan
Keyword(s):  
Green Tea ◽  
Camellia Sinensis ◽  
Health Benefits ◽  
Epigallocatechin Gallate ◽  
Extraction Methods ◽  
Major Constituent ◽  
Epicatechin Gallate ◽  
Wide Range ◽  
Green Tea Catechin ◽  
Anti Inflammatory

Background: Tea (Camellia sinensis, Theaceae) is the second most consumed beverage in the world. Green tea is the least processed and thus contain rich antioxidant level, and believed to have most of the health benefits. </p><p> Methods: We commenced to search bibliographic collection of peer reviewed research articles and review articles to meet the objective of this study. </p><p> Results: From this study, we found that the tea beverage contains catechins are believed to have a wide range of health benefits which includes neuroprotective, anti-inflammatory, antiulcer, antiviral, antibacterial, and anti-parasitic effects. The four major catechin compounds of green tea are epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG), of which EGCG is the major constituent and representing 50-80% of the total catechin content. And also contain xanthine derivatives such as caffeine, theophylline, and theobromine, and the glutamide derivative theanine. It also contains many nutritional components, such as vitamin E, vitamin C, fluoride, and potassium. We sum up the various green tea phytoconstituents, extraction methods, and its medicinal applications. </p><p> Conclusion: In this review article, we have summarized the pharmacological importance of green tea catechin which includes antioxidant potential, anti-inflammatory, antimicrobial, anticancer, antidiabetic and cosmetic application.

scholarly journals A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins

Agronomy ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 107
Author(s):  
Mahmudur Rahman ◽  
Lei Liu ◽  
Bronwyn J. Barkla
Keyword(s):  
Seed Protein ◽  
Storage Proteins ◽  
Protein Extraction ◽  
Seed Storage ◽  
Seed Storage Proteins ◽  
Protein Yield ◽  
Tissue Type ◽  
Single Seed ◽  
Material Optimization ◽  
Hazardous Chemical

Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of Brassica rapa R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

scholarly journals A Structurally Variable Hinged Tetrahedron Framework from DNA Origami

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
David M. Smith ◽  
Verena Schüller ◽  
Carsten Forthmann ◽  
Robert Schreiber ◽  
Philip Tinnefeld ◽  
...  
Keyword(s):  
Self Assembly ◽  
Gel Electrophoresis ◽  
Imaging Techniques ◽  
Building Blocks ◽  
Dna Origami ◽  
Microscopy Technique ◽  
Wire Frame ◽  
Wide Range ◽  
Potential Applications ◽  
Linker Molecules

Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

Comparison of Two Protein Extraction Methods for Tea Residues

2011 ◽  
Vol 138-139 ◽  
pp. 933-936 ◽  
Author(s):  
Xuan Chen ◽  
Hong Yu Luo ◽  
Jun Yu ◽  
Peng Xiang Yue ◽  
Lin Zhou ◽  
...  
Keyword(s):  
High Temperature ◽  
Protein Extraction ◽  
Ethanol Concentration ◽  
Extraction Methods ◽  
Extraction Yield ◽  
Factorial Experiments ◽  
Extraction Technology ◽  
Digestion Method ◽  
Solid Ratio ◽  
Lower Temperature

Alcohol-alkali method and base digestion method were investigated to extract proteins from tea residues, respectively. According to single factorial experiments, results showed that the optimal extraction technology of alcohol-alkali method were pH 12, temperature of 80 °C, ethanol concentration of 60%, liquid-solid ratio of 40, 60 min, and the protein extraction rate reached 15.0%. And the optimal extract conditions of base digestion were pH 12, temperature of 80 °C, liquid-solid ratio of 50, 80 min, which made the protein yield reached 31.5%. Furthermore, alcohol-alkali method was more beneficial to protein extraction from tea residues under lower temperature and weak alkali condition (40-60 °C, pH 8-10). While base digestion had higher extraction yield under high temperature and strong alkali condition (60-80 °C, pH 11-12).

Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens.

1982 ◽  
Vol 28 (4) ◽  
pp. 908-914 ◽  
Author(s):  
R P Tracy ◽  
R M Currie ◽  
D S Young
Keyword(s):  
Quality Assurance ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Two Dimensional ◽  
Protein Loss ◽  
Serum Samples ◽  
Silver Stain ◽  
Two Dimensional Gel Electrophoresis ◽  
Report Quality ◽  
Wide Range

Abstract Currently we are using two different ISO-DALT two-dimensional gel electrophoresis systems, designated MC-Iso 1 and MC-Iso 2, for the analysis of serum and plasma samples. Here we report quality-assurance data for both of these systems. CV values for the slopes of the pH gradient (ISO dimension) are 5.6% of less; CV values for the slopes of the molecular-mass curves (log Mr vs relative mobility in the DALT dimension) are 3.4% or less. We examined the various steps of the analysis in detail for reproducibility and protein loss, using radiolabeled albumin, alpha 2-macroglobulin, and beta 2-microglobulin. Generally, in the first dimension, less protein enters the MC-Iso 2 gels (our routine system in which silver stain is used) than enters the MC-Iso 1 gels (our wide-range system for myeloma serum samples, in which the gel is stained with Coomassie Blue), on the average, 87% as much. The CV at this stage for both systems is 5--8%. During equilibration, considerable amounts of protein are lost (approximately 30% in 10 min) from the ISO gel, and the reproducibility is also decreased. Resolution in the DALT dimension has, in most cases, little or no effect on either recovery or reproducibility. Overall, for most proteins expected to appear in an ISO gel of a given pH range, approximately 50--60% of the starting material may be expected to reside in the sodium dodecyl sulfate slab gel, under our conditions. The two most important variables affecting recovery are the concentration of the NaOH (used as catholyte) and the pH of the starting sample. The overall CV for the process is between 8 and 12%.

scholarly journals Evaluation of effective protein extraction procedure to profile petiole of Moringa oleifera

2020 ◽  
Vol 7 (2) ◽  
pp. 214
Author(s):  
Zetty Amirah Zulkifli ◽  
Zaidah Rahmat
Keyword(s):  
Moringa Oleifera ◽  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Electrophoretic Pattern ◽  
Antioxidant Property ◽  
Extraction Methods ◽  
Protein Profiling ◽  
Sds Page

Moringa oleifera is widely known as multipurpose tree since all of its parts confer multiple functions. The leaf is highly favourable among consumers while the petiole is mostly wasted. There are numerous studies on the flavonoid and antioxidant property of the stem and twig. However, study on the petiole has never been done. There-upon, this study was conducted to develop protein profiling of the petiole. In this study, 6 different protein extraction methods were tested on the fresh petiole before its protein quantity and quality were checked via Bradford assay and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) respectively. The in-solution digestion was then done prior to LC-MS/MS analysis. The protein electrophoretic pattern from the SDS-PAGE proves that method 6 using Tris HCl buffer with incorporation of dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) confers the best quality of protein. It produced the highest number of visible individual bands compared to other methods. Meanwhile, 93 proteins were successfully identified via LCMS analysis where the protein, signal response and carbohydrate metabolism categories confer the highest percentage. High quality and content of the protein extracted from the petiole including the antioxidant, anticancer and antidiabetic protein identified suggested that consuming this part of the plant could enhance nutrients of human body.

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