Showing papers on "Bradford protein assay published in 2015"

Characterization of the Proteins in Honey

Abstract: This study focused on the extraction and identification of honey proteins from selected samples (Acacia, Tualang, and Gelam) from Malaysia. The extraction methods, dialysis, ammonium sulfate, and sodium tungstate precipitation methods, were used to obtain the proteins from the honey. The method of membrane dialysis provided the highest yield of proteins based on the Bradford assay. This method also provided the highest number of protein bands and the clearest protein bands on a 12% polyacrylamide gel compared with the other two precipitation methods. The results revealed that the precipitation methods may degrade the proteins because of the use of strong chemicals and losses after repeated transfers. Mass spectrometric data showed that the honey contained major royal jelly proteins (MRJP) such as MRJP-1, MRJP-2, MRJP-5, and MRJP-7, as well as a few uncharacterized proteins from Apis mellifera. MRJP-1 was the most abundant protein, particularly in Acacia samples. Honey proteins were also shown to possess s...

The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles

Abstract: Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein.

An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

Abstract: Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein–reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 μg mL−1 and 400 μg mL−1 for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 μg mL−1 (R2 = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R2 > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes.

Glomalin-related soil protein in French temperate forest soils: interference in the Bradford assay caused by co-extracted humic substances

Abstract: Summary Thermostable soil protein, known as glomalin, is an important component of soil carbon stocks. Thought to originate from endomycorrhizal fungi, Glomales, this operationally-defined fraction of soil organic matter contains proteins of diverse origin as well as non-protein material, including humic substances. Accumulation results from the balance between production/release and subsequent degradation. Quantification of the protein is subject to uncertainty because of the co-extraction of other components that interfere with the Bradford assay. We studied 10 topsoils from French temperate forests, taken from the national forest monitoring network (Renecofor). Two fractions were extracted, easily extractable (EE) at neutral pH and total extractable (T) at pH 8. Protein was quantified with the colorimetric Bradford method, either by direct calibration using bovine serum albumin (BSA) or by extrapolation of the standard addition plot of BSA. Solubilized organic matter was characterized by using absorbance at 465 and 665 nm and by three-dimensional fluorescence excitation-emission spectroscopy. Neither soil properties nor forest cover influenced glomalin-related soil protein (GRSP) content. Direct assay gave the GRSPEE to be about 1 g kg−1 soil, and GRSPT in the range 3–10 g kg−1, accounting for about 2% of soil organic carbon and about 15% of soil nitrogen. Standard addition plots indicated a two to sixfold under-estimation of protein in total extracts, caused by negative interference with the Bradford assay. The GRSPEE was correlated significantly with both estimates of GRSPT. Under-estimation of GRSPT by direct assay was not related to the E4:E6 ratio but was correlated significantly with the intensity of absorbance at either 460 or 660 nm and with one of the fluorescence peaks. We conclude that GRSPEE is not necessarily more recent than GRSPT and that both fractions may be probes of protein content, but that absolute contents may be under-estimated because of co-extracted humic substances.

Determination of Protein Concentration Using Bradford Microplate Protein Quantification Assay

Abstract: Background: Bradford protein assay is popular due to its ease of performance and relative sensitivity. Many researchers and laboratories in Iran use standard assay of Bradford by cuvette. No commercial kit was available for Bradford microplate assay in Iran. Meanwhile, imported Bradford commercial kits are very expensive and have a long delivery time in Iran. Till now no study or document on Bradford microplate protein quantification assay was reported in Iran, so this study aimed to design and carried out this assay. Methods: In the current study, antigen B of hydatid cyst fluid was used as sample and the assay was performed in microplate wells. The absorbance values were measured at 595 nm and standard curve was generated by Microsoft Office Excel software. The protein concentration of sample was calculated using the equation of the standard curve. Results: Average protein concentration of the sample was 1175 ;mug/ml. The total time needed for reading of absorbance was two minutes approximately. Conclusion: Bradford microplate protein assay is a fast and suitable method. This method could be replacing the time consuming method with cuvette. In addition, if this assay produces as a low price kit it could have many benefits for students and laboratories that need to determine protein concentration by Bradford assay.

Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide.

Abstract: Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol–ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.

Determination of the sensitivity range of biuret test for undergraduate biochemistry experiments

Abstract: Quantitation of the total protein content in a sample is a critical step in protein analysis. Molecular UV-Vis absorption spectroscopy is very efficient in quantitative analysis such as protein quantitation and has extensive applications in chemical and clinical laboratories worldwide. Among the various techniques for protein assay, biuret test is of particular interest in this study. This study sought to verify the sensitivity of biuret assay to protein samples with concentrations ranging from 0.0100 to 5.00 mg/mL. Albumin chicken egg was used as the protein sample. The assay was assessed to determine the range of concentration in which it will show linearity. The biuret test exhibited linearity in a wide range of concentrations (0.1000 ± 0.0004 to 5.00 ± 0.02 mg/mL). The starting protein concentration range established for the calibration curve of this assay is lower than the found literature value (1 mg/sample). The study provides a more workable range of biuret test.

Dairy manure protein analysis using UV-vis based on the Bradford method

Abstract: Organic nitrogen in the form of protein and degraded protein fragments is one of the most important components of dairy manure. Conventionally, protein concentration in dairy manure was estimated by crude protein but it is often overestimated because crude protein contains non-protein nitrogen (NPN). The Bradford assay is predominantly used in measuring clinic samples. In this paper, dairy manure protein assays including the Bradford assay, total Kjeldahl nitrogen method and amino acid analysis were compared using various types of manure samples with a wide range of solids contents at different on-farm treatment steps. The manure protein concentration assay by UV-vis using Bradford method was validated to be compatible to measure the protein concentration in manure samples. Relationships for estimating manure total protein concentration from soluble protein based on the solids contents have been determined. The method described in this study could be used as a supplement method for traditional protein assay, especially when the solids content in manure samples is low. In addition, this method can be easily adapted at a farm level to monitor manure soluble protein and estimate crude protein concentration in order to evaluate the effects of diet change and to manage manure field application amount as fertilizer.

Protein Corona Formation on Magnetite Nanoparticles: Effects of Culture Medium Composition, and Its Consequences on Superparamagnetic Nanoparticle Cytotoxicity.

Abstract: The physicochemical properties and potential cytotoxicity of nanoparticles (NPs) are significantly influenced by their inter- action with proteins, which results in corona formation. Here, we have determined whether corona formation, resulting from interactions between superparamagnetic iron oxide nanoparticles (SPIONs) and different cell culture media, may have consequences for driving NP toxic effects. To address this issue, complementary methods were used. The deter- mination of the hydrodynamic size distribution by ζ (zeta) potential measurement indicated that SPIONs were negatively charged under all conditions but that the actual charge was differed with the cell culture medium used. In vitro protein adsorption studies were carried out using the Bradford protein assay and Fourier transform infrared spectroscopy (FTIR). The Bradford assay revealed that the concentration of unadsorbed proteins and other biomolecules decreased when the SPION concentration increased. FTIR showed that the proteins were, indeed, adsorbed onto the NP surface. This was followed by matrix-assisted laser desorption/ionization time-of-flight secondary ion mass spectrometry (MALDI TOF-SIMS), to identify the adsorbed proteins. Ultimately, three different cell viability assays led to the conclusion that the SPIONs were not toxic for all the concentrations used here. In summary, we found that corona formation on the SPIONs depends on the composition of the culture media but has no consequence for nanotoxicity. We have shown that the application of complementary methods has provided novel insights into SPION/protein interactions.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

Abstract: Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function—an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

Abstract: Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

Advantages of the KDS/BCA Assay over the Bradford Assay for Protein Quantification in White Wine and Grape Juice

Abstract: The present study compared the performance of two colorimetric protein assays, the Bradford and the potassium dodecyl sulfate/bicinchoninic acid (KDS/BCA) assays, for use in wine and grape juice analysis. The Bradford assay was affected by protein type, whereas the KDS/BCA assay had lower protein-to-protein variation. Bovine serum albumin and lysozyme yielded an absorbance (562 nm) vs. protein concentration slope (dose–response curve) similar to that of wine proteins. In the Bradford assay, the presence of 12% ethanol and 200 mg/L of wine polyphenols decreased the protein absorbance by 28 and 16%, respectively, whereas in the KDS/BCA assay such interference was not significant. Among 64 white wines, the correlation between protein haze potential, determined by a heat test, and protein content was better for the KDS/BCA assay. This study confirmed the superiority of the KDS/BCA assay over the Bradford assay for quantifying protein in white grape juice and wine, and it yielded better predictive value with respect to the risk of white wine protein instability.

Fractionation and evaluation of proteins in roots of Echinacea purpurea (L.) Moench

Abstract: Echinacea purpurea (L.) Moench, a member of the Asteraceae family, is a plant rich in flavonoids, essential oils, phenolic compounds, saponins, polysaccharides and glycoproteins. The aim of the study was to evaluate the protein content in dried roots of Echinacea purpurea (L.) Moench after homogenization of roots with liquid nitrogen, extraction in 0.01 mol L-1 phosphate-buffered saline (PBS) and purification followed by fractionation of proteins using gel filtration chromatography. Total concentration of proteins was measured using the Bradford method, and evaluation of the molecular mass of proteins was accomplished by applying the SDS-PAGE gel electrophoresis. The Bradford assay revealed that the highest concentration of proteins in fractions collected after gel filtration chomatography was 4.66-6.07 mg mL-1. Glycoproteins, alkamides and polysaccharides in roots of Echinacea purpurea (L.) Moench are chemical compounds that are responsible for their immunomodulatory properties. However, information about the difference of protein contents in fresh and dried roots of E. purpurea is insufficient.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

Abstract: Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Comparative analysis of the spectrophotometric methods of the protein amount determination in the pectic polysaccharide samples

Abstract: To analyze the reliability of determining the weight fraction of protein in the samples of pectic polysaccharides by the optical density of solutions in the ultraviolet and visible regions of the spectrum, a comparison of eleven methods: Flores, Lowry, Bradford, Sedmac, Ruhemann (ninhydrin reaction) methods, ultraviolet spectrophotometry, methods with Benedict reagent, with Nessler reagent, with amide black, with bicinchoninate reagent, and the biuret method has been carried out. As a result, the insufficient sensitivity of seven of the listed methods has been found, which does not recommend them for these purposes. The Lowry, Bradford, Sedmac methods, and the method with the Nessler reagent can be used for the determination of protein content in the samples of pectic polysaccharides, and the Bradford method can be recommended for characterization of samples of pectic polysaccharides if the weight fraction of protein does not exceed 15%, and the Lowry method can be used for the samples with the weight fraction of protein greater than 15%.

A Bradford multiplexing method for protein estimation in fermented foods: Soy sauces

Abstract: Proteins for foods, in addition to providing nutrition, should also possess specific functional properties that facilitate processing and serve as the basis of product performance. Soy protein is a major component of the diet of food and is increasingly important in the human diet. Hence, here in the present article, we are focusing a rapid and easy method for quantitative determination of total protein content with multiplex samples in any food products such as soy sauce or other traditional fermented foods. We described a bioassay procedure (Bradford method) for the evaluation of total protein content in foods. This method involves measurement of the protein efficiency ratio under standardized conditions. The experiment will provide researchers a scientific way to determine pretentious quality of variety of foods and/or health supplements. Video Clips of Methodology: Requirement and sample preparation method: 3 min 26 sec Full Screen Alternate Assay procedure: 6 min 4 sec Full Screen Alternate Measurement of absorbance using an ELISA microtiter plate reader: 5 min 1 sec Full Screen Alternate

Evaluation and Comparison of Four Protein Extraction Protocols for Mono- and Two-Dimensional Electrophoresis in Mytilus Galloprovincialis

Abstract: In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.

Hydroxyapatite as a support in protease immobilization process

Abstract: Hydroxyapatite is used as a matrix for immobilization of protease from Aspergillus oryzae by a process of adsorption. The matrix obtained has the surface area of 26 m 2 /g and particles in the shape of flakes of diameters no greater than 650 nm. The efficiency of the proposed method was confirmed by the Fourier transform infrared spectroscopy, elemental analysis and by analysis of parameters of the pore structure of matrix and products after immobilization. On the basis of the Bradford method it was found that the greatest amount of enzyme (132 mg/g) was immobilized from a solution of initial enzyme concentration of 7 mg/cm 3 after 24 h of the process.

Selective Modification of Hydrophobic Paper Using a Surfactant for Protein Assay in Urine

Abstract: We used a surfactant to fabricate a microfluidic paper-based analytical device (µPAD) by selective modification of hydrophobic filter paper. Hydrophobic tails of the surfactant penetrate and remain...

In vitro Antifungal, Antioxidant and Cytotoxic Activities of a Partially Purified Protein Fraction from Atlantia monophylla Linn (Rutaceae) Leaf

Abstract: Purpose : To determine the in vitro antifungal and antioxidant activities of the aqueous extract and protein fraction of Atlantia monophylla Linn (Rutaceae) leaf. Methods : Ammonium sulphate (0 – 80 %) precipitation method was used to extract protein from the leaves of A. monophylla Linn (Rutaceae). In vitro antifungal assays were performed by disc-diffusion and micro-broth dilution methods. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2), superoxide and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities were performed to evaluate in vitro antioxidant activities. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was employed to assess the molecular weight of the protein fractions. Protein concentration was determined by Bradford method. The cytotoxicity of these extracts was tested on Vero cell lines. Results: Both the aqueous extract and protein fraction (AMP III) of Atlantia monophylla leaf exhibited higher antifungal activity on Candida albicans than on Aspergillus fumigatus. AMP III fraction showed greater in vitro antioxidant activity than the aqueous extract. SDS-PAGE analyses revealed the presence of two protein bands with molecular weight approximately of 16 and 67 KDa in AMP III. Protein concentration was 240 μg/ml in the aqueous extract and 670 μg/ml in AMP III fraction. The aqueous extract and protein fraction exhibited cytotoxicity at concentrations > 100 μg/ml on Vero cells. Conclusion : Plant-derived proteins/peptides possessing antifungal and antioxidant properties would be a good alternative preparation for the treatment of infectious diseases. Keywords: Atlantia monophylla , Antifungal, Antioxidant, Cytotoxicity, Proteins/peptides, Vero cell lines