The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Manuela Murariu - One of the best experts on this subject based on the ideXlab platform.
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Ultrasound-based Protein Determination in maize seeds.
Ultrasonics sonochemistry, 2015Co-Authors: Gabi Drochioiu, Catalina Ionica Ciobanu, Sabina Bancila, Laura Ion, Brindusa Alina Petre, Claudia Andries, Robert Gradinaru, Manuela MurariuAbstract:The need for a simple and accurate method for Protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total Proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total Proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the Protein content was found to be the key parameter of the improved biuret-based Protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in Protein Determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.
Alfred Weber - One of the best experts on this subject based on the ideXlab platform.
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Rediscovery and Revival of Analytical Refractometry for Protein Determination: Recombining Simplicity With Accuracy in the Digital Era
Journal of pharmaceutical sciences, 2016Co-Authors: Heinz Anderle, Alfred WeberAbstract:Among "vintage" methods of Protein Determination, quantitative analytical refractometry has received far less attention than well-established pharmacopoeial techniques based on Protein nitrogen content, such as Dumas combustion (1831) and Kjeldahl digestion (1883). Protein Determination by quantitative refractometry dates back to 1903 and has been extensively investigated and characterized in the following 30 years, but has since vanished into a few niche applications that may not require the degree of accuracy and precision essential for pharmaceutical analysis. However, because high-resolution and precision digital refractometers have replaced manual instruments, reducing time and resource consumption, the method appears particularly attractive from an economic, ergonomic, and environmental viewpoint. The sample solution can be measured without dilution or other preparation procedures than the separation of the Protein-free matrix by ultrafiltration, which might even be omitted for a constant matrix and excipient composition.
Solange I. Mussatto - One of the best experts on this subject based on the ideXlab platform.
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Interference of some aqueous two-phase system phase-forming components in Protein Determination by the Bradford method.
Analytical biochemistry, 2011Co-Authors: Sara C. Silvério, Sérgio Moreira, Adriane M. F. Milagres, Eugénia A. Macedo, José A. Teixeira, Solange I. MussattoAbstract:*Aqueous Two-Phase Systems (ATPS) are obtained by mixing two aqueous solutions of different constituents that become immiscible under certain critical conditions, like temperature, concentration, etc. Both phases are composed mainly by water (>80%) and each one is enriched in a different component [1]. ATPS formed by two polymers or a polymer and a salt represent the traditional systems. Nevertheless, other alternative biphasic systems can be obtained using surfactants, micellar compounds or ionic liquids. Due to the high percentage of water present in their composition, ATPS can provide a gentle environment for the extraction and recovery of sensitive biological materials, such as Proteins. Different methodologies can be used for Protein quantification, such as UV absorbance spectroscopy (280 nm), chemical methods (like Kjeldahl) or colorimetric Determination based on dye binding assays (like Bradford, BCA or Lowry). Among them, the Bradford assay is the most currently used for Protein quantification in ATPS due to its simplicity, sensitivity and fast response. This assay is based on the absorbance shift observed when the dye Coomassie® Brilliant Blue G-250 binds to Protein. The peak absorbance of the acidic solution of Coomassie® changes from 465 to 595 nm when binding occurs. The binding process is fast (5 minutes) and the Protein-dye complex formed is stable for about 1 hour [2]. The use of a single reactive and the sensitivity of the dye to small amounts of Protein make the Bradford method widely used for Protein Determination. However, the method can suffer significant interference from some compounds, like detergents and alkaline agents. It is also known that the presence of some salts under certain concentration can compromise the method response. Nevertheless, poor information is found in the literature about interference of specific phase-forming components of ATPS. In this work, the interference in the Bradford method caused by several salts and polymers usually used in ATPS formation was investigated. For that purpose, calibration curves were obtained for bovine serum albumin (BSA) in the presence of different concentrations of sulfates (Na2SO4, (NH4)2SO4, MgSO4 and LiSO4), phosphates (Na2HPO4, NaH2PO4, K2HPO4 and KH2PO4), citrates (citric acid and trisodium citrate), acetates (acetic acid and sodium acetate) and polymers (PES Mw=100000, UCON Mw=3900, Ficoll Mw=70000 and PVP Mw=40000). Comparison of these curves with that obtained for BSA in distilled water, allow us to conclude that salts produce a more significant effect in BSA calibration curve than polymers. In all cases, a reduction in the absorbance at 595 nm (A595) was observed. For most of the salts tested, reduction ≥20% was obtained in A595 when a concentration ≥1% (w/w) was present. The only exceptions were observed for the salts NaH2PO4, KH2PO4, citric acid and acetic acid that, probably due to their acidic pH, presented reductions in the A595 lower than 12% for 1% (w/w). For sulfates, the change of the cation also provided a change in the calibration curve and (NH4)2SO4 showed the higher interference (A595 reduction of 25% for a concentration of 1% (w/w)). Fig. 1 shows the reduction in A595 in the presence of different (NH4)2SO4 concentrations.
Klaus Becker - One of the best experts on this subject based on the ideXlab platform.
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A modified dot-blot method of Protein Determination applied in the tannin-Protein precipitation assay to facilitate the evaluation of tannin activity in animal feeds.
The British journal of nutrition, 2002Co-Authors: E. Hoffmann, S. Muetzel, Klaus BeckerAbstract:Tannins have received considerable attention from animal nutritionists as potential agents for modifying ruminal fermentation patterns, or for exploring new feed resources. This group of secondary plant compounds is defined by their ability to form complexes with Proteins. A widely accepted method for assaying the biological activity of extracted tannins is the precipitation of bovine serum albumin. The Protein carries a radioactive label ( 125 I) to allow direct quantification from the precipitate. Tannin–Protein complexes dissolve in sodium dodecylsulfate solution. A dot-blot assay for Protein Determination, which is based on the reversible binding of a fluorochrome, benzoxanthene yellow, to the Protein spots and is not disturbed by the presence of detergents, can replace the radioactive method by a fluorimetric measurement. A novel alternative to the last part of the dot-blot assay is to scan the stained Protein spots in situ using a video camera and computer image analysis. Several filter sets were tested and, within a concentration range of 0·1–2·0 mg Protein/ml, each of them yielded results identical to the original method while the time required was only 30 % of the working time consumed by the original procedure. The modified dot-blot assay should be applicable to the evaluation of tannin activity in all shrub and tree foliages considered as animal feed.
Gabi Drochioiu - One of the best experts on this subject based on the ideXlab platform.
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Ultrasound-based Protein Determination in maize seeds.
Ultrasonics sonochemistry, 2015Co-Authors: Gabi Drochioiu, Catalina Ionica Ciobanu, Sabina Bancila, Laura Ion, Brindusa Alina Petre, Claudia Andries, Robert Gradinaru, Manuela MurariuAbstract:The need for a simple and accurate method for Protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total Proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total Proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the Protein content was found to be the key parameter of the improved biuret-based Protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in Protein Determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.
