Showing papers on "Bradford protein assay published in 2019"
TL;DR: The results revealed that protein adsorption on PLGA- andPLGA-PEG-NP didn't depend on NP size within the range of 100 and 200 nm, and depletion of proteins which are involved in immune response was remarkable and indicated a prolonged circulation time in body.
80 citations
TL;DR: Investigating the protein corona that forms around poly(lactide-co-glycolide) (PLGA) NPs at different serum concentrations using two substantially different serum types revealed that the physiological environment critically affects the protein adsorption on PLGA NPs with significant impact on the NP–cell interaction.
Abstract: Background: When nanoparticles (NPs) are applied into a biological fluid, such as blood, proteins bind rapidly to their surface forming a so-called "protein corona". These proteins are strongly attached to the NP surface and confers them a new biological identity that is crucial for the biological response in terms of body biodistribution, cellular uptake, and toxicity. The corona is dynamic in nature and it is well known that the composition varies in dependence of the physicochemical properties of the NPs. In the present study we investigated the protein corona that forms around poly(lactide-co-glycolide) (PLGA) NPs at different serum concentrations using two substantially different serum types, namely fetal bovine serum (FBS) and human serum. The corona was characterized by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), Bradford protein assay, zeta potential measurements, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Additionally, the time-dependent cell interaction of PLGA NPs in the absence or presence of a preformed protein corona was assessed by in vitro incubation experiments with the human liver cancer cell line HepG2. Results: Our data revealed that the physiological environment critically affects the protein adsorption on PLGA NPs with significant impact on the NP-cell interaction. Under comparable conditions the protein amount forming the protein corona depends on the serum type used and the serum concentration. On PLGA NPs incubated with either FBS or human serum a clear difference in qualitative corona protein composition was identified by SDS-PAGE and LC-MS/MS in combination with bioinformatic protein classification. In the case of human serum a considerable change in corona composition was observed leading to a concentration-dependent desorption of abundant proteins in conjunction with an adsorption of high-affinity proteins with lower abundance. Cell incubation experiments revealed that the respective corona composition showed significant influence on the resulting nanoparticle-cell interaction. Conclusion: Controlling protein corona formation is still a challenging task and our data highlight the need for a rational future experimental design in order to enable a prediction of the corona formation on nanoparticle surfaces and, therefore, the resulting biodistribution in the body.
63 citations
TL;DR: In this paper, the authors investigated the source of interference in the Bradford quantification of Glomalin-related soil protein and proposed several methodological improvements based on identified interferences, and found that the extent of interference varied between different soils.
Abstract: Glomalin‐related soil protein (GRSP), an operationally defined fraction of soil organic matter containing protein and various other components, is usually quantified using the colorimetric nonspecific Bradford method. This method is limited by a short working range, a nonlinear response and interference from co‐extracted compounds. These limitations hinder the exact quantification of the protein component. The aim of this study was to investigate the source of interference in the Bradford quantification of GRSP and propose several methodological improvements based on identified interferences. The easily extractable and total GRSP in five topsoils with contrasting texture, organic carbon content and land use were compared. Results showed that: (a) the extent of interference varied between different soils, (b) the standard addition method overestimated the extent of inhibition, (c) absorbance should be corrected for colour, (d) use of the ratio of absorbances at 595 and 465 nm, A₅₉₅/A₄₆₅, is not recommended because it is sensitive to pH and dilution‐dependent absorbance at 465 nm, (e) although a quadratic fit to the protein calibration curve was better than the linear fit, it was not possible for the dilution method, and (f) estimation of protein content from the dilution curve of the soil extract appeared to be suitable as it integrates the often observed, and hitherto unexplained, effect of dilution on the calculated protein content of soil extracts and avoids artefacts because of the choice of protein spike and dilution. HIGHLIGHTS: Soil protein colorimetric quantification is hampered by co‐extracted compounds. Variants of Bradford assay of glomalin‐related soil protein are tested for five contrasting soils. Direct assay underestimates soil protein, but standard addition may overestimate. Controlled sample dilution with colour correction might give the best estimate of soil protein.
26 citations
TL;DR: The utility of 2,2,2-trichloroethanol-based protein quantification is demonstrated and the protein visualization in polyacrylamide gels based on 2,7 μg low-volume assay enabled visualization of proteins following SDS-PAGE.
Abstract: The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.
23 citations
TL;DR: The development of 3D‐μPADs and 3D-μTPADs can further facilitate the use of these platforms for colorimetric bioassays and show good correlation between the theoretical and actual concentrations.
Abstract: In this paper we describe the fabrication of novel 3D microfluidic paper-based analytical devices (3D-μPADs) and a 3D microfluidic thread/paper-based analytical device (3D-μTPAD) to detect glucose and BSA through colorimetric assays. The 3D-μPAD and 3D-μTPAD consisted of three (wax, heat pressed wax-printed paper, single-sided tape) and four (hole-punched single-sided tape, blank chromatography circles, heat-pressed wax-printed paper, hole-punched single-sided tape containing trifurcated thread) layers, respectively. The saturation curves for each assay were generated for all platforms. For the glucose assay, a solution of glucose oxidase (GOx), horseradish peroxidase, and potassium iodide was flowed through each platform and, upon contact with glucose, generated a yellow-brown color indicative of the oxidation of iodide to iodine. For the protein assay, BSA was flowed through each device and, upon contact with citrate buffer and tetrabromophenol blue, resulted in a color change from yellow to blue. The devices were dried, scanned, and analyzed yielding a correlation between either yellow intensity and glucose concentration or cyan intensity and BSA concentration. A similar glucose assay, using unknown concentrations of glucose in artificial urine, was conducted and, when compared to the saturation curve, showed good correlation between the theoretical and actual concentrations (percent differences <10%). The development of 3D-μPADs and 3D-μTPADs can further facilitate the use of these platforms for colorimetric bioassays.
21 citations
TL;DR: This work functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and conducted a comprehensive comparative study of their properties essential to applications in biosensing.
Abstract: The surface functionalization of magnetic nanoparticles improves their physicochemical properties and applicability in biomedicine. Natural polymers, including proteins, are prospective coatings capable of increasing the stability, biocompatibility, and transverse relaxivity (r2) of magnetic nanoparticles. In this work, we functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and we conducted a comprehensive comparative study of their properties essential to applications in biosensing. First, we examined the influence of environmental parameters on the size of prepared nanoclusters and synthesized protein-coated nanoclusters with a tunable size. Second, we showed that protein coating does not significantly influence the r2 relaxivity of clustered nanoparticles; however, the uniform distribution of individual nanoparticles inside the protein coating facilitates increased relaxivity. Third, we demonstrated the applicability of the obtained nanoclusters in biosensing by the development of a nuclear-magnetic-resonance-based immunoassay for the quantification of antibodies against tetanus toxoid. Fourth, the protein coronas of nanoclusters were studied using SDS-PAGE and Bradford protein assay. Finally, we compared the colloidal stability at various pH values and ionic strengths and in relevant complex media (i.e., blood serum, plasma, milk, juice, beer, and red wine), as well as the heat stability, resistance to proteolytic digestion, and shelf-life of protein-coated nanoclusters.
18 citations
TL;DR: An inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants and its performance is more robust and accurate over time and the respective concentration range.
Abstract: Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants. The reliability and reproducibility of the method are tested in a long-term study and compared with lysis quantification via the DNA measurement. The results show that its performance is more robust and accurate over time and the respective concentration range. Additionally, both methods are used for cell lysis process monitoring in a recombinant Chinese hamster ovary fed-batch process. In the presented process, by applying the established assay, the lysis rate k DL is determined to be constant over time at 4.6 × 10 -4 lysed cell concentration (LCC) per TCC and time (LCC/TCC/h). In contrast, DNA data did not confirm the constant lysis rate due to variations of the content per cell during cultivation. Thus, information on the RPC can facilitate the determination of the optimal harvest time point with respect to purity and in improving process characterization.
11 citations
TL;DR: A novel protein assay applying the 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (BisANS) fluorescent dye has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients.
Abstract: In many biology- and chemistry-related research fields and experiments the quantification of the peptide and/or protein concentration in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different experiments beyond or instead of the well-known standard protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This molecule has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold diluted samples) concentrations (from 0.28 up to more than 100 μg/mL). The BisANS protein assay is valid and applicable for quantification of the amount of protein in different biological and/or chemical samples.
11 citations
TL;DR: Results show that lysis buffer not only causes background interference but also affects the protein–dye chromogenic process and the sensitivity of the Bradford assay to proteins in lysis buffers was increased, and the interference delivered by lysisbuffer was considerably reduced.
Abstract: The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.
8 citations
TL;DR: To study the release patterns of protein bovine serum albumin (BSA), porous poly(ε-caprolactone)-chitosan scaffolds with entrapped BSA were fabricated by using supercritical CO2 for its potential use in tissue engineering applications.
Abstract: To study the release patterns of protein bovine serum albumin (BSA), porous poly(e-caprolactone)-chitosan scaffolds with entrapped BSA were fabricated by using supercritical CO 2 (scCO 2 ) for its potential use in tissue engineering applications. An emulsion, consisting of a polymer-solvent solution and buffer protein solution was saturated with scCO 2 at 12 MPa and 37 °C and then rapidly depressurized through a release valve causing bubble nucleation and precipitation of the composite material. The controlled total protein release from biodegradable poly(e-caprolactone) with 5% chitosan (w/w) scaffolds was assessed by Bradford protein assay. After 16 to 20 days of protein release testing, 58.8% of the protein was released from composite with PCL (M w =10,000 g/mol) and 43.9% from composite with PCL (M w =60,000 g/mol). Preliminary studies for characterization of the prepared composite biomaterials using FTIR spectra, ESEM photo analysis and DSC analysis have been carried out.
7 citations
TL;DR: It is suggested that rapid diagnosis of immunoglobulins is possible using the concept of protein corona formation ex vivo, and it is predicted that the changes in secondary structure of primary corona determine the overall signature of surface binding proteins in PC.
TL;DR: In this paper, the authors used ultrathin silicon nanoporous membranes (SNMs) combined with detection using ultraviolet absorption for continuous and real-time monitoring of biomolecules.
Abstract: The increase in demand for continuous and real-time monitoring of permeation of biomolecules is addressed by using highly selective ultrathin silicon nanoporous membranes (SNMs) combined with detection using ultraviolet absorption. The membranes, with an average pore size of 8.8 nm, are fabricated using semiconductor batch processes including chemical vapor deposition and rapid thermal annealing. Bovine serum albumin (BSA) of a concentration of $250~\mu \text{g}$ /ml is used as the test molecule. The concentration of BSA diffused through the membrane is measured using optical transduction based in-house developed sensor. The photocurrent obtained from the sensor is measured every 15 min and compared with the standard Bradford assay at the same time-stamp. The concentration estimated by the sensor is found to agree with the Bradford assay with a standard deviation of 1.4%. The throughput of the membrane is increased by fabricating an array of SNMs, which showed an increase in diffusion rate by 3.8 times with respect to the single SNM. The clogging of pores by the biomolecules is analyzed using ionic conductivity experiments. The structural integrity of BSA diffused through the SNM is also analyzed.
TL;DR: The combined use of cyclodextrins with a commercial Bradford reagent concentrate allows to determine protein concentrations in a Laemmli-type buffer, containing both SDS and reducers, in a single step (without any precipitation) with a simple spectrophotometric assay.
Abstract: Determination of protein concentrations prior to (sodium dodecyl sulfate) SDS electrophoresis is made difficult by the simultaneous presence of SDS and reducers in the buffers used for protein extraction. Reducers interfere with the copper-based assays, while SDS interferes with the dye-binding assays. The combined use of cyclodextrins with a commercial Bradford reagent concentrate, described in this chapter, allows to determine protein concentrations in a Laemmli-type buffer, containing both SDS and reducers, in a single step (without any precipitation) with a simple spectrophotometric assay. The use of various cyclodextrins brings compatibility not only with SDS but also with other nonionic and ionic detergents such as sodium deoxycholate or detergents of the Triton type.
Patent•
11 Jan 2019
TL;DR: In this article, a differential protein detection method based on 5-phenyl-1-(p-methylphenyl)-1H-triazole and serum action was proposed.
Abstract: The invention relates to a differential protein detection method based on 5-phenyl-1-(p-methylphenyl)-1H-triazole and serum action. The differential protein detection method comprises the following steps: mixing a serum sample with a 5-phenyl-1-(p-methylphenyl)-1H-1,2,3-triazole compound; removing high-abundant protein; measuring the concentration of protein by a Bradford method; carrying out a bidirectional electrophoresis experiment; and processing a film image and authenticating a mass spectrum. The invention further provides a preparation method of the 5-phenyl-1-(p-methylphenyl)-1H-triazole.
TL;DR: The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.
Abstract: BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guerin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro.
METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg// ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays.
RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10.
CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.
01 Mar 2019
TL;DR: In this article, the feasibility of neutron irradiation as a method for reducing milk allergens was analyzed and the results strongly indicate that the allergenicity of milk proteins can be reduced by fast neutron irradiations in an inverse exponential manner.
Abstract: This experiment was conducted to analyze the feasibility of neutron irradiation as a method for reducing milk allergens. In a temperature-controlled environment, whole milk was irradiated with 4 MeV fast neutron irradiation from a 5 Ci Pu-Be source. After irradiation, the samples were treated with Bradford reagent as an assay. The final concentrations from the Bradford Assay indicated protein degradation rather than denaturation leading to further testing with discontinuous polyacrylamide gel electrophoresis. The gel electrophoresis results determined that most proteins had been degraded into unidentifiable proteins components. The remaining main allergenic proteins, alpha-casein and beta-lactoglobulin, decreased by a factor of 3 and 13 respectively after 30 hours of irradiation in an inverse exponential manner. The results strongly indicate that the allergenicity of milk allergens can be reduced by fast neutron irradiation.
Patent•
11 Jan 2019
TL;DR: In this paper, a method for detecting differential proteins in the serum interacted with 1-(4-chlorophenyl)-5-phenyl-1H-1,2,3-triazole was proposed.
Abstract: The invention relates to a method for detecting differential proteins in the serum interacted with 1-(4-chlorophenyl)-5-phenyl-1H-1,2,3-triazole, comprising the following steps of: mixing a serum sample with 1-(4-chlorophenyl)-5-phenyl-1H-1,2,3-triazole compound; removing the high-abundance protein; quantitatively determining the protein concentration by the Bradford method; carrying out the two-dimensional electrophoresis; and carrying out the film image processing, the mass spectrum identification and the like The invention further provides a preparation method of the 1-(4-chlorophenyl)-5-phenyl-1H-1,2,3-triazole
Patent•
11 Jan 2019
TL;DR: In this paper, a differential protein detection method based on 1-phenyl-5-(p-methylphenyl)-1H-triazole and serum action was proposed.
Abstract: The invention relates to a differential protein detection method based on 1-phenyl-5-(p-methylphenyl)-1H-triazole and serum action. The differential protein detection method comprises the following steps: mixing a serum sample with a 1-phenyl-5-(p-methylphenyl)-1H-1,2,3-triazole compound; removing high-abundant protein; measuring the concentration of protein by a Bradford method; carrying out a bidirectional electrophoresis experiment; and processing a film image and authenticating a mass spectrum. The invention further provides a preparation method of the 1-phenyl-5-(p-methylphenyl)-1H-1,2,3-triazole.
Patent•
11 Jan 2019
TL;DR: In this article, the authors proposed a differential protein detection method through interaction between 5-(4methoxyl phenyl)-1-phenyl-1H-triazole and serum.
Abstract: The invention relates to a differential protein detection method through interaction between 5-(4-methoxyl phenyl)-1-phenyl-1H-triazole and serum. The method comprises the following steps: mixing a serum sample with a 5-(4-methoxyl phenyl)-1-phenyl-1H-1, 2, 3-triazole compound; removing high-abundant protein; determining protein concentration through a Bradford method; performing a bidirectional electrophoresis experiment; performing film image processing and mass spectrum identification; and so on. The invention also provides a preparation method of the 5-(4-methoxyl phenyl)-1-phenyl-1H-1, 2,3-triazole.