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With the development of techniques for rapid microbial identification, MALDI-TOF MS has become an important tool for clinical identification of fungi. Problems such as the applicability and standardization of protein extraction methods have hindered the development of MALDI-TOF MS technology in the fungal field. This paper analyzed the complex structure of fungal cell walls, introduced the protein extraction methods recommended by MALDI-TOF MS commercial mass spectrometry systems, discussed the protein extraction methods for the identification of various genera of yeast-like fungi and filamentous fungi by MALDI-TOF MS, such as direct smear method, formic acid acetonitrile extraction method and magnetic bead grinding method, and summarized the current status and drawbacks of protein extraction methods in fungal identification by MALDI-TOF MS with a view to providing theoretical reference for subsequent research.
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Objective To explore protein extraction efficiency from formaldehyde-fixed paraffin embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue samples with different protocols. Methods Six different lysis buffers with 100 °C or 105 °C. treatments were used for protein extraction, followed by evaluation of protein quantity and quality with Bradford, sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, Western blotting and immunohistochemistry (IHC), using 8 FFPE samples of ESCC. Results The optimal method for protein extraction from FFPE ESCC tissue was Laemmli lysis buffer (Buffer 4) treated with 100 °C incubation, evidenced by highest amount of protein recovery. Western blotting and IHC method measured consistent 14-3-3σ expression in FFPE ESCC tissue samples. Protein precipitated by two volumes of acetonitrite acetonitrile(ACN) (0.1% trifluoroacetic acid) relative to protein amount reduced background staining on SDS-PAGE gels by commassie staining. Conclusion Laemmli lysis buffer combined with 100 °C incubation has the highest protein extraction efficiency from FFPE ESCC tissue samples for Western blotting measurement of protein biomarkers, and ACN protein precipitation can further eliminate residual cross- linked protein by FFPE.
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ABSTRACT Biologically active proteins isolated from plant species can be used in traditional medicine as prolific resources for new drugs Morinda pubescens Sm., Rubiaceae, is a promising medicinal plant which is widely used in folk medicine to treat fever due to primary complex, ulcer and glandular swellings. In this study, proteins were extracted from the leaves of M. pubescens, and precipitated with ammonium sulphate at various saturation concentrations ranging from 20 to 80%. The precipitated protein sample obtained with 80% saturation was further purified using ultrafiltration membrane (<10 kDa). SDS-PAGE analysis identified the presence of crude and ultrafiltered protein bands. FTIR spectrum of the ultrafiltered protein fractions depicted the presence of hydroxyl and carbonyl groups of proteins. The ultrafiltered proteins exhibited increased cytotoxic activity on A549 cells at the concentrations ranging from 15 to 100 µg/ml. About 98% cell viability was also observed in Vero cells treated with the maximum concentration of 100 µg/ml of ultrafiltered protein extract. DNA fragmentation was observed in A549 cells treated with 10 µg/ml of ultrafiltered proteins, indicating the onset of apoptosis.
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OBJECTIVE:To establish the technical system that is suitable for protein extracting in Angelicae sinensis seed and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and provide technical support for detecting the protein quality and variety purity. METHODS:Using protein content and number of electrophoretic bands as indexes,8 methods,includ-ing voncentrated gel method,salt-soluble protein method,electrode buffer method,dimercaptosyl alcohol (DTT) method,urea method,mercaptoethanol method,trimethylolaminomethane (Tris) method,and acetone precipitation method,were conducted to extract the protein in A. sinensis seed and screen the optimal extraction method. Then based on optimal extraction method,effects of different materials-lipid ratios,sample dilution times(sample volume)and separate gel concentration on SDS-PAGE were investi-gated. RESULTS:Mercaptoethanol method extracted the highest protein contents(29.931 mg/g),with many electrophoretic bands and clear background. When mercaptoethanol method was used as optimal extraction method,electrophoresis effects were the best in the conditions of materials-lipid ratio of 1:10,sample volume of 5 times,separate gel concentration of 15%,which obtained 18 bands totally. CONCLUSIONS:Established protein extraction method and SDS-PAGE technical system are suitable for detecting the purity of A. sinensis seed.
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Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.
Subject(s)
Acetates/analogs & derivatives , Electrophoresis/methods , Musa/chemistry , Phenylacetates , Plant Proteins/isolation & purification , Plant Roots/enzymology , /methodsABSTRACT
Objective To compare optimal protein extraction method of velvet antler ( VA ) and explore the repairmen activity of velvet antler proteins ( VAPs ) on 1-methyl-4-phenylpyridinium ( MPP +) damaged nerve cell.Methods VAPs was obtained from fresh velvet antler by homogenization and micronization method respectively.The protein content of VAPs was measured by Bradford and the molecular weight was identified by SDS-PAGE.Scanning electron microscope (SEM) was perfomed to observe the ultrastructure of VAPs.Neuroblastoma cell line (SH-SY5Y) damage was induced by MPP +, and activity of each compound was compared by the proliferation of damaged cell detected by MTT method.Results The yield of micronization method of VAPs was higher than homogenization method and with better characters both in SDS-PAGE and SEM.Activity detection indicated that VAPs at the concentration of 125 μg/mL by homogenate method could significantly increase the proliferation rate of damaged SH-SY5Y cells.By contrast, VAPs by micronization method had more effective effect at 62.5μg/mL, and proliferation rate could reach 25.45% .Conclusion Micronization method does not only acquire more protein but also has cell proliferation activity, is a relatively ideal way of protein extraction of velvet antler.
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Objective To compare the separation effects of protein samples extracted by two different methods with two -dimensional gel electrophoresis (2-DE) .Methods Ultrasonic disruption and glass-beads grounding were used to prepare protein samples of Gram-negative bacteria , Gram-positive bacteria and animal tissues .The actual results of the two sample preparation methods were compared by 2-DE.Results The 2-DE maps of samples extracted by the two methods were obtained.Conclusion The 2-DE maps of glass-beads grounding samples are better than those of ultrasonic disruption thanks to their lower backgrounds , which are beneficial for further image analyses .
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This study was aimed to investigate active ingredients from A mp e lop s is me galop hylla of protein expression by using two-dimensional gel electrophoresis (2-DE). Methods: isn this study, proteinsfrom the tender leaves of A mpelopsis megalophylla were precipitated by Tris-phenol extraction and ammonium acetate methanol precipitation. Then using 2-DE technology to isolate protein.Finally, 2-DE was analyzed by the scanner, and got protein characteristic maps. Result:According to the differences in ratio of gray value, the seven differential protein points were chosen to identify the mass spectrum. At present, two proteins were identified, namely the hypothetical protein and the unnamed protein product. Conclusion: the application of 2-DE technology for A mpelopsis megalophyllathe active ingredient of protein expression at the level of molecular research laid a good foundation.
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Aims: Find a suitable method for the protein extraction from flower buds of Solanum lycopersicum. Study Design: Compare some kinds of protein extraction methods and find the best one among them suitable to tomato flower buds. Place and Duration of Study: Biological Science and Technology College, between June 2010 and July 2011. Methodology: The proteins for electrophoresis were extracted using different methods, such as trichloroacetic acid /acetone (TCA/acetone), Sodium dodecyl sulfate (SDS), Trissaturated phenol (Tris-Phen), Phenol/SDS and Direct lysis method. After silver staining, different patterns of protein spots were observed in the gels. Results: Few spots were found by SDS and Phenol/SDS extractions, more spots by immediate dissolution but the most impurities, less protein productivity though more spots by Tris-Phen extractions, and more protein productivity and better apart effect by TCA/acetone. The 2-DE image background was the clear and the protein spots were the most by TCA/acetone method. Conclusion: TCA/acetone method is much more suitable as extraction method for protein two-dimensional electrophoresis of tomato flower buds.