Method For Determination Of Protein Research Articles

Soluble Protein and Amino Acid Content Affects the Foam Quality of Sparkling Wine.

Proteins and amino acids are known to influence the foam characteristics of sparkling wines. However, it is unclear to what extent they promote foam formation and/or stability. This study aimed to investigate the effect of protein content and amino acid composition, measured via the bicinchoninic acid assay and high-performance liquid chromatography, respectively, on the foaming properties of 28 sparkling white wines, made by different production methods. Foam volume and stability were determined using a robotic pourer and computer vision algorithms. Modifications were applied to the protein determination method involving the use of yeast invertase as a standard in order to improve quantification accuracy. The protein content was found to be significantly correlated to parameters representative of foam stability, as were the amino acids arginine, asparagine, histidine, and tyrosine. Additionally, the production method was found to influence the foam collar height, which favored foaming in Méthode Traditionnelle wines over other those made by production methods. Understanding the contributions of key wine constituents to the visual and mouthfeel parameters of sparkling wine will enable more efficient production of high-quality wines.

Compared diagnostic value of proteinuria definition applying different methods at pregnants with preeclampsia

Colorimetric method of protein determination in urine with use of pyrogallol red (PGR) has certain advantages over a routine method of proteinuria determination with use of sulfosalicylic acid (SSA) which is still applied in most clinics in the Russian Federation. For the purpose to determinate the diagnostic significance of proteinuria estimation by different methods at pregnants with preeclampsia, conducted a comparative research of proteinuria levels at 68 pregnants with preeclampsia and at 15 healthy pregnant by two methods: with sulfosalicylic acid and with pyrogallol red. At the same patients the diagnostic significance of protein/creatinine ratio was evaluated when using the method with PGR. It was revealed that proteinuria determination with PGR- method had authentically higher sensitivity, and proteinuria indices when using this method corresponds to the criteria for evaluation of preeclampsia severity specified in the Ministry of Health Russian Federation Order No. 572n 2012 and the Clinical Recomandations “Hypertensia during pregnancy. Preeclampsia. Eclampsia” (2012). In case of proteinuria determination by method with SSK it is necessary to consider lower sensitivity of the method and adhere to earlier classification of preeclampsia (gestosis) where proteinuria more 1 g/l was regarded as the symptom of severe preeclampsia. Confirmation of the diagnostic significance of protein/creatinine ratio needs further researches.

CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.

新しい尿中たんぱく定量法の検討(その 2) : トリクロール酢酸-ポンソー S 改良法(界面活性剤の添加)

新しい尿中たんぱく定量法の検討(その 2) : トリクロール酢酸-ポンソー S 改良法(界面活性剤の添加)

Evaluation of Spectrophotometric Methods for Determination of Proteins in Capsular Polysaccharide of Haemophilus influenzae Type b (PRP)

Evaluation of Spectrophotometric Methods for Determination of Proteins in Capsular Polysaccharide of Haemophilus influenzae Type b (PRP)

The effect of late autumn cutting of lucerne (Medicago sativa L.) on the growth rhythm of the first cut in the following year

Abstract The field experiment aimed at determination of influence of autumn harvest on rhythm of the growth of lucerne (Medicago sativa L.) and yield quality of the first cut in the following year was carried out in Spodnji Gabernik (258 m above sea level, 46°15′54.94″ N and 15°34′6.05″ E), in the fourth growth year of the lucerne variety ‘Soča’. The experiment was designed as a randomized block in four replications. The individual treatments represented weekly autumn cuttings (1) 24/9/2014, (2) 01/10/2014, (3) 08/10/2014, (4) 15/10/2014 and (5) 22/10/2014. The paper presents data associated with the amount of lucerne dry matter yield (DMY) per treatment compared with the DMY of the first spring cut in the year 2015 (18/05/2015). The growth rhythm of lucerne in each treatment, which took place before the first cut in year 2015, was based on the measurements of the height of the plants. The yield quality was evaluated by NIRS method for determination of crude protein (CP), the net energy of lactation (NEL) and metabolic energy (ME) in dry samples regarding to DMY of each treatment. In autumn 2014, statistically significant higher dry matter yields were obtained in later harvested treatments (5 and 4). Similarly, in spring 2015 the trend of higher crop dry matter yield was observed in treatments harvested later in the autumn of 2014. However, the differences were not significant. Statistically significant differences between treatments were acquired within CPY, NELY and MEY. Before the first mowing, in spring 2015, statistically significant trend of higher plants was also detected in treatments with later cutting in 2014.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Serum proteins and their diagnostic utility in veterinary medicine: a review

The evaluation of serum proteins and their electrophoretic pattern is a well-established laboratory method in the diagnosis of many diseases in humans, which has replaced the biochemical determination of the concentrations of albumin and the ratio of albumin to globulins. The measurement of serum proteins may be an important diagnostic tool for the detection, diagnosis, and monitoring of various diseases and pathological processes. The results of serum protein electrophoresis can be one of the most useful diagnostic aids in a wide spectrum of diseases, including infectious and inflammatory diseases, renal or gastrointestinal disorders, immunodeficiency states, as well as paraproteinaemias caused by lymphoid or plasma cell neoplasia. Although many studies have been carried out to determine the usefulness of the determination of serum proteins and their electrophoretic pattern in various disease conditions and disorders also in animals, serum protein evaluation is still a relatively little-used diagnostic tool in veterinary medicine. In this article, methods of serum protein determination, their diagnostic utility in animal care practice and their different patterns in dysproteinaemias and paraproteinaemias are reviewed.

Raman Spectroscopy as a Real-Time In Situ Analyzer for Cell Culture Bioprocesses

The development of fast and reliable methods for protein determination are of great relevance to a diversity of areas from industry to diagnostics. Molecular Imprinted Materials (MIM) has proved to be an interesting methodology for protein analysis however further studies of the effect of the experimental parameters and starting materials in the performance of the MIM are still required. Caffeic acid (CAF) is employed for the first time as a monomer to tailor a synthetic receptor for a protein target. This was done by bulk-electropolymerization, applying a constant potential of +2.0 V, for 30 s, on a carbon screen-printed electrode, immersed in a solution of protein and CAF prepared in phosphate buffer. Annexin A3 (ANXA3) was selected as protein target due to the fact that this is an emerging biomarker in prostate cancer.The assembly of the protein imprinted material (PIM) was followed by Electrochemical Impedance Spectroscopy (EIS) and Raman Spectroscopy. A non-imprinted material (NIM) was prepared in parallel as control.Square wave voltammetry (SWV) was used to monitor the electrochemical signal of the [Fe(CN)6]3−/[Fe(CN)6]4− redox for the quantification of ANXA3.The optimized PIM-based device showed average detection limits (LOD) of 0.095 ng/mL, a linear behavior against log (concentration) between 0.10, and 200 ng/mL and good selectivity. The NIM-based device showed random behavior against protein concentration. Finally, the PIM-sensor was successfully applied to the analysis of ANXA3 in spiked urine samples.

Rediscovery and Revival of Analytical Refractometry for Protein Determination: Recombining Simplicity With Accuracy in the Digital Era

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.

Protein Imprinted Material electrochemical sensor for determination of Annexin A3 in biological samples

The development of fast and reliable methods for protein determination are of great relevance to a diversity of areas from industry to diagnostics. Molecular Imprinted Materials (MIM) has proved to be an interesting methodology for protein analysis however further studies of the effect of the experimental parameters and starting materials in the performance of the MIM are still required. Caffeic acid (CAF) is employed for the first time as a monomer to tailor a synthetic receptor for a protein target. This was done by bulk-electropolymerization, applying a constant potential of +2.0V, for 30s, on a carbon screen-printed electrode, immersed in a solution of protein and CAF prepared in phosphate buffer. Annexin A3 (ANXA3) was selected as protein target due to the fact that this is an emerging biomarker in prostate cancer.The assembly of the protein imprinted material (PIM) was followed by Electrochemical Impedance Spectroscopy (EIS) and Raman Spectroscopy. A non-imprinted material (NIM) was prepared in parallel as control.Square wave voltammetry (SWV) was used to monitor the electrochemical signal of the [Fe(CN)6]3−/[Fe(CN)6]4− redox for the quantification of ANXA3.The optimized PIM-based device showed average detection limits (LOD) of 0.095ng/mL, a linear behavior against log (concentration) between 0.10, and 200ng/mL and good selectivity. The NIM-based device showed random behavior against protein concentration. Finally, the PIM-sensor was successfully applied to the analysis of ANXA3 in spiked urine samples.

The protein content of seaweeds: a universal nitrogen-to-protein conversion factor of five

A global drive to source additional and sustainable biomass for the production of protein has resulted in a renewed interest in the protein content of seaweeds. However, to determine accurately the potential of seaweeds as a source of protein requires reliable quantitative methods. This article systematically analysed the literature to assess the approaches and methods of protein determination and to provide an evidence-based conversion factor for nitrogen to protein that is specific to seaweeds. Almost 95 % of studies on seaweeds determined protein either by direct extraction procedures (42 % of all studies) or by applying an indirect nitrogen-to-protein conversion factor of 6.25 (52 % of all studies), with the latter as the most widely used method in the last 6 years. Meta-analysis of the true protein content, defined as the sum of the proteomic amino acids, demonstrated that direct extraction procedures underestimated protein content by 33 %, while the most commonly used indirect nitrogen-to-protein conversion factor of 6.25 over-estimated protein content by 43 %. We therefore determined whether a single nitrogen-to-protein conversion factor could be used for seaweeds and evaluated how robust this would be by analysing the variation in this factor for 103 species across 44 studies that span three phyla, multiple geographic regions and a range of nitrogen contents. An overall median nitrogen-to-protein conversion factor of 4.97 was established and an overall mean nitrogen-to-protein conversion factor of 4.76. We propose that the overall median value of 5 be used as the most accurate universal seaweed nitrogen-to-protein (SNP) conversion factor.

Review of methods for determination of total protein and peptide concentration in biological samples.

Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit-for-use, quality-by-design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

Spectrophotometric methods to determine protein concentration.

Measuring the concentration of proteins is an essential part of enzyme evaluations or to monitor protein yields during protein isolation procedures. Decisions on the usefulness of any isolation procedure depend on knowing the relative concentrations of a particular protein or enzyme in relation to the concentrations of all the proteins present. Protein concentration in solution is generally measured with spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein. This review describes protein determination methods for measuring protein concentration in solution.

Development of a new method for determination of total haem protein in fish muscle

Using classic haem protein quantification methods, the extraction step in buffer or acid acetone often becomes limiting if muscle is oxidised and/or stored; haem-proteins then tend to bind to muscle components like myofibrils and/or biomembranes. The objective of this study was to develop a new haem protein determination method for fish muscle overcoming such extractability problems. The principle was to homogenise and heat samples in an SDS-containing phosphate buffer to dissolve major muscle components and convert ferrous/ferric haem proteins to hemichromes with a unique absorption peak at 535nm.Hb-recovery tests with the new and classic methods showed that the new method and Hornsey’s method performed significantly better on fresh Hb-enriched cod mince than Brown’s and Drabkin’s methods; recovery was ⩾98%. However, in highly oxidised samples and in cod protein isolates made with acid pH-shift processing, the new method performed better than Hornsey’s method (63% and 87% vs. 50% and 68% recovery). Further, the new method performed well in fish muscle with ⩽30% lipid, <5% NaCl and pH 5.5–7.0; it was also unaffected by freezing/frozen storage.

LINEARIZAÇÃO DA METODOLOGIA DE BRADFORD PARA APLICAÇÃO NA QUANTIFICAÇÃO DAS PROTEÍNAS TOTAIS DO LEITE BOVINO POR ESPECTROFOTOMETRIA NO UV-VIS

Reliable methods for determination and quantification of total protein in food are essential information to ensure quality and safety of food trade. The objective of this study was to evaluate the linearity of calibration curves obtained from different proteins (blood serum albumin-BSA, α-LA, β-LG, αs, β and κ-CAS) with the reagent of Bradford. Comercial UHT skimmed bovine milk was analyzed for the determination of total protein using the Bradford method by reading at 595 nm. The determination of the concentrations of total milk protein was achieved by linear regression. The Bradford method showed a high sensitivity for the determination of total proteins in bovine milk dilution 1:25 to values closer to those obtained by the Kjeldahl method. The results showed that the calibration curve of standard proteins β-CN and BSA obtained better linearity with less variation in the absorbance measurements for the determination of total protein of milk.

Sequence Based Prediction of DNA-Binding Proteins Based on Hybrid Feature Selection Using Random Forest and Gaussian Naïve Bayes

Developing an efficient method for determination of the DNA-binding proteins, due to their vital roles in gene regulation, is becoming highly desired since it would be invaluable to advance our understanding of protein functions. In this study, we proposed a new method for the prediction of the DNA-binding proteins, by performing the feature rank using random forest and the wrapper-based feature selection using forward best-first search strategy. The features comprise information from primary sequence, predicted secondary structure, predicted relative solvent accessibility, and position specific scoring matrix. The proposed method, called DBPPred, used Gaussian naïve Bayes as the underlying classifier since it outperformed five other classifiers, including decision tree, logistic regression, k-nearest neighbor, support vector machine with polynomial kernel, and support vector machine with radial basis function. As a result, the proposed DBPPred yields the highest average accuracy of 0.791 and average MCC of 0.583 according to the five-fold cross validation with ten runs on the training benchmark dataset PDB594. Subsequently, blind tests on the independent dataset PDB186 by the proposed model trained on the entire PDB594 dataset and by other five existing methods (including iDNA-Prot, DNA-Prot, DNAbinder, DNABIND and DBD-Threader) were performed, resulting in that the proposed DBPPred yielded the highest accuracy of 0.769, MCC of 0.538, and AUC of 0.790. The independent tests performed by the proposed DBPPred on completely a large non-DNA binding protein dataset and two RNA binding protein datasets also showed improved or comparable quality when compared with the relevant prediction methods. Moreover, we observed that majority of the selected features by the proposed method are statistically significantly different between the mean feature values of the DNA-binding and the non DNA-binding proteins. All of the experimental results indicate that the proposed DBPPred can be an alternative perspective predictor for large-scale determination of DNA-binding proteins.

Development and Validation of HPLC-UV Method for Determination of Bovine Serum Albumin and Myoglobin Proteins

A simple HPLC with UV detection method is developed and validated for determination of Bovine Serum Albumin and Myoglobin proteins in a standard solution of the two protein as well as in a plasma spiked with theses two proteins.Separation was achieved on a reversed-phase C18, 5.0 µm, 150mm ◊ 4.6 mm inner diameter column using a mobile phase consisting ofsolution A (900 mL of water, 100 mL Acetonitrile, 10 mL Trifluroacetic acid) and solution B (900 mL of ace tonitrile, 100 mL water, 10 mL Trifluroacetic acid) with gradient elution of 5 to 50% of solution B in 15 minutes . This new method is validated in accordance with requirements for new methods, which include accuracy, precision, selectivity, linearity and r ange, robustness, limit of detection and limit of quantitation. The method demonstrates good linearity over the range of 1 -1000 ppm for the two proteins with r 2 greater than 0.998.The percentage recovery of the method is within 97.9 to 102.0% for the twoproteins. Precision of this method reflected by relative standard deviation of the area of six replicate injections of each protein at five concentration levels (1.0, 100.0, 300.0, 500, and 1000.0 ppm) was found to be less than 2%. Limit of quantitation ofthe two proteins is low which enables the determination of these proteins at low concentration. OriginalResearch Article

Development of Method for Determination of Lead, Iodine, and Protein Content in Fish Sauce

The aim of this investigation was to develop a method for the determina- tion of the lead, iodine, and protein content in fish sauce. For statistical data, five samples of fish sauce that cost 9.0-43.0 Bahts were randomly bought from a supermarket in Chiang Mai Province. Five replicates of each sample were digested and measured for the lead amount, using absorbance by atomic ab- sorption spectroscopy, and each replication was injected six times. The iodine content were measured from the five samples, and each sample was investi - gated in five replications using the iodimetric method. The protein contents was determined by applying the Kjedahl method using the five samples. The lead determination method was accepted because of the statistical data. Using fish sauce as a seasoning agent is a practice that is safe from lead toxicity because the lead contents were found to be lower than the stipulated Thai Food Act Limit which is 1.0 ppm. The determination method for iodine content needs to be further developed by controlling the pH of the reaction medium to be neutral or to be that of a dilute acid. The protein determination method needs to be investigated for more statistical data. Consumers should also pay better attention to the sample label because it was observed in this investigation that there was a highly priced sample contained low nutritional values of protein besides showing the wrong label.

Sensitive Determination of Proteins with Naphthol Green B by Resonance Light Scattering Technique

A new quantitative determination method for trace proteins using naphthol green B (NGB) by resonance light scattering (RLS) spectroscopy has been developed. The method is based on the interaction of protein and NGB at pH 3.00, which causes a substantial enhancement of the resonance scattering signal of NGB in the wavelength range 300–550 nm with the maximum RLS at 392.0 nm. Under optimum conditions, the linear range is 0.010–28.2 μg/ml for bovine serum albumin (BSA) and 0.010–31.3 μg/ml for human serum albumin (HSA). The detection limits (S/N=3) are 8.2 ng/ml for BSA and 7.9 ng/ml for HSA, respectively. There is little or no interference from amino acids, most of the metal ions, or other coexisting substances. The easy-to-use method, with high sensitivity and good reproducibility, was satisfactorily applied to the determination of total protein in human serum samples. The determination results for human serum samples are identical to those provided by clinical physicians.