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Showing papers on "Bradford protein assay published in 2011"


Journal ArticleDOI
20 Mar 2011
TL;DR: The Bradford protein assay is used to measure the concentration of total protein in a sample and actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex.
Abstract: [Abstract] The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents (i.e., DTT and beta—mercaptoethanol) and metal chelators (i.e., EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding. This technique was invented by Bradford (1976).

116 citations


Journal ArticleDOI
TL;DR: It is shown that at a given temperature, increasing the extrusion moisture content resulted in a slight increase in the overall protein water solubility, averaging approximately 5% per 10% increase in moisture content.

100 citations


Journal ArticleDOI
TL;DR: The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595nm) can be identified and corrected by recording absorption spectra in the region of 350-850mm.

90 citations


Journal ArticleDOI
TL;DR: It is demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.
Abstract: Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and β-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.

57 citations


Journal ArticleDOI
14 Mar 2011-Analyst
TL;DR: This report assesses how the protein incubated in sample tubes may be lost due to adsorption and a model system to evaluate these phenomena is proposed.
Abstract: The non-specific loss of protein analytes can have a major effect on assay results particularly where the concentrations of such analytes are extremely low and the matrix is complex. This report assesses how the protein incubated in sample tubes may be lost due to adsorption. Use of proteins, such as bovine serum albumin (BSA), may be used to pre-treat tubes to reduce such losses. However, such losses may also be associated with structural perturbations leading to changes in immunogenicity (as a result of alterations in specific epitope-related conformations). This can lead to erroneous results or lack of comparability with a range of methodologies such as the bicinchoninic protein assay and immunoassays or when surface plasmon resonance (SPR)-based approaches are used. A model system to evaluate these phenomena is proposed.

48 citations


Journal ArticleDOI
05 Mar 2011

41 citations


Journal ArticleDOI
TL;DR: Hetero-polysaccharide mucilage was extracted from the seed coats of different citrus rootstocks viz. Rough lemon, Sachtion citrumelo and Yuma citrange for investigating its biochemical and molecular properties.

38 citations


Book ChapterDOI
TL;DR: This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations.
Abstract: This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered to be an important component of biocompatibility screens. The products of lipid peroxidation, lipid hydroperoxides, and aldehydes, such as MDA, can be measured via a thiobarbituric acid reactive substances (TBARS) assay. In this assay, which can be performed in cell culture or in cell lysate, MDA combines with thiobarbituric acid (TBA) to form a fluorescent adduct that can be detected at an excitation wavelength of 530 nm and an emission wavelength of 550 nm. The results are then expressed as MDA equivalents, normalized to total cellular protein (determined by Bradford assay).

34 citations


Journal ArticleDOI
TL;DR: The influence of high polyethylene glycol and phosphate salt concentrations on the readings of three colorimetric protein assays: Bradford, DC (Bio-Rad) and ninhydrin assay is investigated, which displays minimal protein-to-protein variation and high sensitivity.

30 citations


Journal ArticleDOI
TL;DR: In this paper, the differences in the emulsifying properties of isolated soy protein prepared in the pilot plant (heated and spray dried) or in the laboratory from the same soy flakes were determined.
Abstract: The purpose of this study was to determine the differences in the emulsifying properties of isolated soy protein prepared in the pilot plant (heated and spray dried) or in the laboratory (unheated and freeze-dried), from the same soy flakes. When the thermal transitions were measured by micro-calorimetry, the protein isolated in the pilot plant showed a very broad thermal transition, while the native isolate showed two distinct transition peaks, attributed to β-conglycinin and glycinin denaturation. Electrophoretic analysis and protein assay of the soluble protein in the fractions revealed a significantly smaller amount of protein recovered in the centrifugal supernatant for the isolated soy protein prepared in the pilot plant than for the native protein. A larger amount of ions was recovered in the pilot plant isolate. However, the thermal treatment of the solutions increased the recovery of the pilot plant isolate proteins in the centrifugal supernatant, with an opposite effect for the native soy protein. A significantly larger amount of pilot plant isolated protein was needed to prepare emulsions with the same characteristics of those prepared with native soy protein. The emulsions prepared with pilot plant isolates showed much lower susceptibility to heating than those prepared with native protein.

23 citations


Journal ArticleDOI
TL;DR: The results showed that HPCD induced leakage loss of the proteins and DNA of E. coli as a function of treatment time, and the denaturation was enhanced by increasing treatment time.
Abstract: Protein changes in Escherichia coli, when subjected to high-pressure carbon dioxide (HPCD) at 10 MPa and 3 °C for 5–75 min, were assessed using the Bradford method, 2D electrophoresis (2-DE) and liquid chromatography-electrospray ionization-MS-MS (LC-ESI-MS-MS). The changes in DNA in E. coli under the same conditions were also investigated by using flow cytometry with propidium iodide and acridine orange, agarose gel electrophoresis (AGE) and the comet assay. The results showed that HPCD induced leakage loss of the proteins and DNA of E. coli as a function of treatment time. With regard to the protein changes, 182 proteins in the 2-DE profile were not found in the HPCD-treated E. coli. Among 20 selected protein spots exhibiting significant changes in intensity, 18 protein spots were identified as 15 known proteins and two as hypothetical proteins. These proteins were involved in cell composition, energy metabolism pathways, nucleic acid metabolism, global stress regulation and general metabolism. The DNA denaturation of E. coli induced by HPCD was demonstrated in this study for the first time to our knowledge, and the denaturation was enhanced by increasing treatment time. However, HPCD did not cause DNA degradation, as suggested by both AGE analysis and the comet assay.

Journal ArticleDOI
TL;DR: Substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay is reported, which indicates a similar BCA reaction mechanism for NHS and protein.

Journal ArticleDOI
TL;DR: Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA), however, the combined absorbance of sulfo-nHS and BSA was not strictly additive.

Journal ArticleDOI
TL;DR: Sy synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.
Abstract: Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy)-[2, 2'-bipyrimidine]-4, 6-diyl)bis(1H-pyrazol-3, 1-diyl)) dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375) and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

Journal ArticleDOI
01 Jul 2011
TL;DR: The methods considered in this lecture have multi-year history and applied widely in the laboratory practice, there are some crucial points, which must be taken into consideration while choosing the method permitting reliably and with a high specificity and reproducibility to quantify protein this paper.
Abstract: Protein quantification is an integral part of any investigation related to protein isolation, purification, characterization, and analysis. Although the methods considered in this lecture have multi-year history and applied widely in the laboratory practice, there are some crucial points, which must be taken into consideration while choosing the method permitting reliably and with a high specificity and reproducibility to quantify protein.

Journal ArticleDOI
TL;DR: PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation and should find application in study of cell signaling, including questions related to aging and regeneration.
Abstract: We introduce a fully integrated multistep protein assay that reports both protein identity and size. To report these two properties, a microfluidic design strategy integrates pore limit electrophoresis (PLE) with a heterogeneous immunoassay in a single microchannel (PLE-IA). PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation. In a single-channel multistage assay approach, an antibody to follistatin was first immobilized in a polyacrylamide PLE gradient gel, near the origin of the separation axis. Immobilization relies on pore-limit exclusion of the antibody and not on chemical functionalization of either the sieving matrix or the antibody, making assay customization by an end-user straightforward. Subsequently, target and ladder protein species were electrophoretically introduced into the antibody-patterned PLE channel. Species having an affinity for the immobilized antibody were detected via heterogeneous immunoassay. Noninter...

Dissertation
14 Oct 2011
TL;DR: In this paper, a project aimed to determine the protein profiles and concentration in honeys, effect of storage conditions on the protein content and the interaction between proteins and polyphenols.
Abstract: This project aimed to determine the protein profiles and concentration in honeys, effect of storage conditions on the protein content and the interaction between proteins and polyphenols. Thirteen honeys from different botanical origins were analyzed for their protein profiles using SDS-PAGE, protein concentration and phenolic content, using the Pierce Protein Assay and Folin-Ciocalteau methods, respectively. Protein-polyphenol interactions were analyzed by a combination of the extraction of honeys with solvents of different polarities followed by LCjMS analysis of the obtained fractions. Results demonstrated a different protein content in the tested honeys, with buckwheat honey possessing the highest protein concentration. We have shown that the reduction of proteins during honey storage was caused, partially, by the protein complexation with phenolics. The LCjMS analysis of the peak eluting at retention time of 10 to 14 min demonstrated that these phenolics included flavonoids such as Pinobanksin, Pinobanksin acetate, Apigenin, Kaemferol and Myricetin and also cinnamic acid.

Journal ArticleDOI
TL;DR: It is demonstrated that the fluorescent in situ gene protein assay methodology is capable of resolving gene and protein patterns simultaneously on a cell-by-cell basis.
Abstract: We present a novel methodology combining traditional fluorescent in situ hybridization with an in situ protein detection technology called proximity ligation assay. This method has potential to perform a detailed analysis of the relationship between gene status and corresponding protein expression in cells and tissues. We demonstrate that the fluorescent in situ gene protein assay methodology is capable of resolving gene and protein patterns simultaneously on a cell-by-cell basis.

Journal ArticleDOI
TL;DR: This is the first report of analytical interference by quinolone and quinine derivatives in the PRM assay, and lowest interfering concentrations were mostly above estimated therapeutic concentrations.

Journal ArticleDOI
TL;DR: Olink Bioscience's Proseek protein assay enables sensitive detection and quantification of proteins in a 1-μl sample volume, making it highly suitable for analyzing biomarkers and cytokines in precious biological samples.
Abstract: Olink Bioscience's Proseek protein assay enables sensitive detection and quantification of proteins in a 1-μl sample volume. With the Proseek Assay Development kit, a new assay can rapidly be developed for any target protein with appropriate antibodies available. Proseek's minimal sample consumption and excellent assay performance make it highly suitable for analyzing biomarkers and cytokines in precious biological samples.

Journal ArticleDOI
TL;DR: Four protein extraction methods and three protein quantification techniques were compared with Paenibacillus sp.
Abstract: Four protein extraction methods and three protein quantification techniques were compared with Paenibacillus sp. whole cells. Proteins were extracted using conventional cell disruption techniques encompassing: sonication and glass bead vortexing, as well as BugBuster Master Mix extraction and Total Protein Kit extraction. The Bradford assay, Folin-Lowry assay and UV absorbance at 280 nm were used for protein quantification methods. Differences in protein profiles were examined by 2D-PAGE and subsequently analysed using PDQuest Advanced 2D Analysis software. All extraction methods revealed proteins over broad molecular weight range. UV absorbance at 280 nm using the NanoDrop™1000 and the Bradford assay yielded best quantification results. Rapid and effective disruption and quantification of Paenibacillus sp. strain D9 cells was successfully achieved using the combination of Total Protein Extraction Kit-UV280 followed by BugBuster Master-UV280.

Patent
11 May 2011
TL;DR: In this article, a method for analyzing protein content in 2-keto-L-gulonic acid, which comprises the following steps: adopting an optimized Coomassie brilliant blue quantitative protein method (a Bradford method), preparing phosphate buffer solution, bovine serum protein standard solution, adding the Coomenie Brilliant blue G-250 staining agent, drawing a standard curve according to absorbency added values after the Coomasserie brilliantblue is combined with the protein, and calculating the protein content.
Abstract: The invention relates to a method for analyzing protein content in 2-keto-L-gulonic acid, which comprises the following steps: adopting an optimized Coomassie brilliant blue quantitative protein method (a Bradford method), preparing phosphate buffer solution, bovine serum protein standard solution and Coomassie brilliant blue G-250 staining agent, adding the Coomassie brilliant blue G-250 stainingagent into the bovine serum protein standard solution, drawing a standard curve according to absorbency added values after the Coomassie brilliant blue is combined with the protein, then adding the 2-keto-L-gulonic acid into a Coomassie brilliant blue reagent, and calculating the protein content in the 2-keto-L-gulonic acid according to the absorbency added values and the slope coefficient of a protein concentration standard curve. The analyzing method is simple, convenient and sensitive, has the advantages of good stability, high accuracy, good repeatability and low requirement on detection equipment, is quite suitable for analyzing and detecting the protein content in the 2-keto-L-gulonic acid in the industrial production.

Journal ArticleDOI
TL;DR: The results showed that serum protein adsorption and complement activation were augmented for nanoparticles with a larger size below 400 nm, and nanoparticles in the nanometer and submicrometer range were phagocytosed more readily than either smaller or larger particles.
Abstract: The purpose of this study is to evaluate the effect of particle size on serum protein opsonization and in vitro macrophage uptake of polyethyleneglycol modified poly (D, L-lactide-co-glycolide) nanoparticles (PEG-PLGA-NPs). PEG-PLGA-NPs were prepared by modified-spontaneous emulsification solvent diffusion (modified-SESD) method. Serum protein adsorptions to PEG-PLGA-NPs were evaluated by bicinchoninic acid (BCA) protein assay and enzyme-linked immunosorbent assay (ELISA). Complement activation was also investigated by ELISA for complement fragments iC3b. Uptake of PEG-PLGA-NPs by macrophages was measured by fluorescence spectrometer. The results showed that serum protein adsorption and complement activation were augmented for nanoparticles with a larger size below 400 nm. Phagocytosis of PEG-PLGA-NPs by murine peritoneal macrophages involved serum-independent and serum-dependent phagocytosis. Serum-independent phagocytosis decreased, while serum-dependent phagocytosis increased with the increase of particle size in the nanometer and submicrometer range. Consequently, nanoparticles with size of about 400 nm were phagocytosed more readily than either smaller or larger particles

Journal ArticleDOI
15 Jun 2011-Talanta
TL;DR: Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.

Journal ArticleDOI
TL;DR: The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide to the reporter enzyme, glucose-6-phosphate dehydrogenase and proved effective for detection of a high-molecular-weight model protein tagged with HA.
Abstract: A practical approach for constructing enzyme-multiplied immunoassay technique (EMIT)-based protein/peptide assays is described. Normally used in small-molecule drug testing, EMIT is a homogeneous assay method that is attractive for its simplicity, sensitivity, and rapidity. The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase. The 13-min assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high-molecular-weight model protein tagged with HA. Similar EMIT-based assay approaches may be developed for applications in biotoxin and infectious disease detection.

Journal Article
TL;DR: Changes in culture media can increase the production of recombinant proteins in host bacteria and the presence of nutrients, such as glucose, alone not only can not increase the amount of production but it might even decrease it.
Abstract: Background: Streptokinase is one of the antigenic proteins secreted by streptococcus pyogenes. This protein has an important role in bacterial pathogenesis. The aim of this study was to produce recombinant forms of this enzyme so that the product would change in accordance with changes in the media. Materials and Methods: In this experimental study, we amplified the streptokinase gene by polymerase chain reaction (PCR) method. After extraction, it was sub-cloned to prokaryotic expression vector pET32a. pET32a-Ska was transferred to E.coli BL21-DE3-plySs strain. Protein production was induced by IPTG and optimization of culture media and OD of bacteria. The recombinant protein was extracted by Ni-NTA and its concentration was measured by Bradford assay. Western- Blot analysis was used to verify the recombinant protein. Results: The nucleotide sequence of the amplified gene was the same as streptokinase gene of the streptococcus pyogenes. The production of recombinant streptokinase by induction of plasmid pET32a-Ska was done by IPTG. The recombinant streptokinase had the same antigenic properties as natural streptokinase. The largest amount of recombinant protein was produced in bacteria concentrations with OD = 0.8. Also, the production of the recombinant protein was higher in media with no glucose. Conclusion: Changes in culture media can increase the production of recombinant proteins in host bacteria. The presence of nutrients, such as glucose, alone not only can not increase the amount of production but it might even decrease it.

01 Jan 2011
TL;DR: This work is a study of how the protein secondary structure affects the absorption spectra in the Bradford assay and use of full spectra instead of the prescribed single-wavelength measurements helps to avoid misinterpretations in protein concentration determination.
Abstract: When using proteins in applications the amount of protein is a fundamental property. For enzyme immobilization there is a need to measure the protein concentration at a solid support. The system studied in this work is mesoporous particles. Today immobilized protein content is normally estimated from the residual protein concentration in the solution after immobilization. Any changes to the protein content for example through leakage have to be determined in the same indirect manner. It would be useful with a method to measure the actual immobilized protein content. By direct measurements on the solid material (here porous particles) with proper corrections for the disturbance from the material this could be achieved. The correction is determined by the character of the disturbance. This character can be identified by observations of absorption spectra. Use of full spectra instead of the prescribed single-wavelength measurements helps to avoid misinterpretations in protein concentration determination. This methodology was also adapted to studies of release of therapeutic proteins from drug formulations. Together with the protein polymers from the tablet matrix was released. The presence of these polymers gave rise to different disturbances in the concentration measurements and could be identified and in one case corrected for. In this work is also a study of how the protein secondary structure affects the absorption spectra in the Bradford assay. When Coomassie blue (the dye in the assay) binds to native protein there is an increase in the absorption at ~590 nm. Coomassie blue also binds to amyloid fibrils, fibrous protein structures mainly consisting of β-sheets. When binding to the fibrils the absorption shifts to longer wavelengths. By comparison to shifts caused by solvent polarity and viscosity properties of the amyloid binding sites are suggested.

Journal Article
TL;DR: In this paper, the protein content in Xinmailong injection was measured by Bradford assay connected with standard addition method of second derivative spectrum method to exclude interference, and a common standard curve was established to detect the content present in protein by standard addition methods of SDS method.
Abstract: OBJECTIVE To evaluate the protein content in Xinmailong injectionMETHODS The protein content in Xinmailong injection was measured by Bradford assay connected with standard addition method of second derivative spectrum method to exclude interferenceRESULTS The present study was conducted to elucidate that the protein content of about 015 mg·mL-1 present in Xinmailong injection and a common standard curve to detect the content present in protein by standard addition methods of second derivative spectrum method was establishedCONCLUSION The protein content in Xinmailong injection could be evaluate accurately by the method of Bradford assay connected with standard addition method of second derivative spectrum method

Dissertation
01 May 2011
TL;DR: Protease from coriander leaf (Coriandrum sativum) was evaluated for its ability to detect selected heavy metals using Bradford-protease-casein assay system as mentioned in this paper.
Abstract: Protease from coriander leaf (Coriandrum sativum) was evaluated for its ability to detect selected heavy metals using Bradford-protease-casein assay system. Considering the highly polluted environment with heavy metals contributed by industrial wastages and its implications on public health, this present study was dedicated to provide a rapid and sensitive assay for the detection of heavy metals in the environmental samples. The basis of the protein assay using casein as a substrate relies upon the inability of the Bradford reagent to stain polypeptide with less than molecular weight of 2 kDa. Casein that has been stained by the Bradford reagent gives a dark blue color. However, the degradation product is not stained by the reagent and the solution remains brown in color. In the presence of heavy metals that inhibit protease activity, casein would remain undigested and the color would remain blue even after incubation. Optimization studies were carried out for this protease prior to heavy metals inhibition studies. The optimization studies include enzyme concentration, substrate concentration, pH, temperature and time of incubation. The optimum concentration of protease, substrate, temperature and incubation time for protease were 0.45 mg/ml protease, 0.43 mg/ml casein, 35oC and 20 min respectively after a period of heavy metals incubation. This enzyme was then purified through anion exchanger using DEAE- Cellulose column and gel filtration using Agilent ZORBAX column. The molecular weight detected was around 55 kDa. Protease activity obtained from coriander was found to be optimum at pH around 8 to 9.5. For this bioassay, two heavy metals showed inhibition towards enzyme activity at a concentration of 1 mg/l. The inhibition shown by the heavy metals on protease activity were around 40% for mercury and 70% for zinc. The IC50 values of mercury and zinc were 3.22 mg/l and 0.73 mg/l respectively. The limits of detection (LOD) for mercury and zinc were 0.24 mg/l and 0.23 mg/l respectively. The limits of quantitation (LOQ) for mercury and zinc were 0.80 mg/l and 0.76 mg/l respectively. This bioassay using coriander protease was found not to be sensitive towards pesticides and xenobiotics. The advantage of the protease bioassay compared to other bioassay relies on its rapidity, simplicity, economical value, stability in severe conditions such as pH and temperature as well as relatively interference free from detergents, solvents and pesticides.