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 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 Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

2016 ◽  
Vol 35 (2) ◽  
pp. 100-106 ◽  
Author(s):  
Jameel R. Al-Obaidi ◽  
Noor Baity Saidi ◽  
Siti Rokhiyah Ahmad Usuldin ◽  
Siti Nahdatul Isnaini Said Hussin ◽  
Noornabeela Md Yusoff ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods

scholarly journals Modified TCA/acetone precipitation of plant proteins for proteomic analysis

2018 ◽  
Author(s):  
Liangjie Niu ◽  
Hang Zhang ◽  
Zhaokun Wu ◽  
Yibo Wang ◽  
Hui Liu ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Protein Modification ◽  
Protein Extraction ◽  
Extended Period ◽  
Extraction Methods ◽  
Precipitation Method ◽  
Plant Proteins ◽  
Prolonged Incubation ◽  
Acetone Precipitation ◽  
Modified Method

AbstractProtein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling

Crop Science ◽  
2009 ◽  
Vol 49 (2) ◽  
pp. 395-402 ◽  
Author(s):  
Chengjian Xie ◽  
De Wang ◽  
Xingyong Yang
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Cotton Seedling

Optimized protein extraction methods for proteomic analysis ofRhizoctonia solani

Mycologia ◽  
2008 ◽  
Vol 100 (6) ◽  
pp. 867-875 ◽  
Author(s):  
Dilip K. Lakshman ◽  
Savithiry S. Natarajan ◽  
Sukla Lakshman ◽  
Wesley M. Garrett ◽  
Arun K. Dhar
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods

scholarly journals Evaluation of Protein Extraction Methods for Proteomic Analysis of Non-Model Recalcitrant Plant Tissues

2012 ◽  
Vol 85 (2) ◽  
pp. 177-183 ◽  
Author(s):  
Dubravko Pavoković ◽  
Bojana Križnik ◽  
Marijana Krsnik-Rasol
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Plant Tissues

scholarly journals An efficient protein extraction method applied to mangrove plant Kandelia obovata leaves for proteomic analysis

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Jiao Fei ◽  
You-Shao Wang ◽  
Hao Cheng ◽  
Yu-Bin Su
Keyword(s):  
Proteomic Analysis ◽  
Protein Extraction ◽  
Elongation Factor ◽  
Extraction Methods ◽  
Vital Role ◽  
Kandelia Obovata ◽  
Protein Patterns ◽  
Mangrove Plant ◽  
B Method ◽  
Flight Mass Spectrometry

Abstract Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

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