Showing papers on "Bradford protein assay published in 2013"

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

Abstract: Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

Flow Process for Electroextraction of Total Proteins from Microalgae

Abstract: Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae (Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology.

Comparison of two methods of tear sampling for protein quantification by Bradford method

Abstract: by the Bradford method. Results were analyzed by Student’s t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

Ozone effects on soluble protein content of Acer negundo , Quercus robur and Platanus spp. pollen

Abstract: In this study, the effect of three different ozone (O3) levels on soluble protein content of Acer negundo, Quercus robur and Platanus spp. pollen was investigated. Pollen was directly collected from each plant and exposed to O3 values below, equal and four times the limit for human health protection in an environmental chamber system. With this chamber, it was possible to control and reproduce the O3 levels between different experiments. Total protein content was determined colorimetrically with Coomassie protein assay reagent (Pierce) by the Bradford method. Results showed that O3 has significant effects on pollen causing a decrease in soluble proteins that seem to be dependent on species and gas concentration. Also, the existence of reproducibility in the effects of O3, within the same concentration, on the protein content of pollen was verified.

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model.

Abstract: For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. Specific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Abstract: Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

A New Colorimetric Method for the Determination of Proteins

Abstract: A new colorimetric method for the determination of proteins was investigated using the local dye "Uri isi". Serial dilution of a solution of bovine serum albumin (BSA) was made. Protein assay was carried out photo-metrically with BSA using both the biuret reagent and the local dye. The results showed that the optical density decreased from 1.269 to 0.189 with an increase in the concentration of protein in the case of biuret reagent and also decreased from 0.276 to 0.174 with increase in the concentration of protein in the case of the local dye. The results suggest that the local dye was more sensitive than biuret reagent for assaying proteins since the bovine serum albumin was diluted 100 times before the local dye could be used for the assay.

Nonspecific particle-based method with two-photon excitation detection for sensitive protein quantification and cell counting.

Abstract: A novel easy-to-use homogeneous method utilizing two-photon excitation (TPX) for quantification of proteins or counting of eukaryotic cells in solution has been developed. This highly sensitive technique is based on the adsorption competition between the sample and fluorescently labeled protein to micrometer-sized carboxylate modified polystyrene particles and detection of two-photon excited fluorescence. The adsorption of the labeled protein to the particles was detected as a distinct fluorescence on individual microparticles. Analyte protein or eukaryotic cells interacted with particle surface and reduced the adsorption of labeled protein to the particles resulting in a decrease of the fluorescence. The optimizations of assay conditions were performed separately for protein quantification and cell counting, and the principle of the method was confirmed with the fluorescence microscopy imaging. The protein quantification assay allowed the determination of picogram quantities (1.2 μg/L) of protein, and the cell counting assay allowed three cells in the sample with an average variation of approximately 10% in the signal. The protein assay sensitivity was more than 500-fold improved from the common most sensitive commercial methods. Moreover, the dynamic range of the assay was broad, approximately 4 orders of magnitude. The cell assay has sensitivity comparable to the most sensitive commercial method. The developed method tolerates interfering agents such as neutral detergents found in cell lysate samples even at high concentrations. The method is experimentally fairly simple and allows the expansion for the use of the TPX technology.

Effect of chitosan on protein content in the medium culture of osteoblasts exposed to oxidative stress

Abstract: Chitosan is a derivative of chitin which has potential for use in bone regeneration and has been reported can stimulate bone formation. Oxidative stress as one cause of bone damage, was found increased in osteoporosis, periodontitis and arthritis. One of the species oxygen reactive (ROS), hydrogen peroxide, has been reported can inhibit osteoblast pro-liferation. This study was aimed to investigate the effect of various chitosan concentrations on protein content in the culture medium of human osteoblast-like cell line, MG 63, which was exposed to hydrogen peroxide. MG 63 cells were exposed to various chitosan concentrations (% w/v) 0.1, 0.2, 0.4, and 1.6%. Culture cells without chitosan were used as a control. Cells were growth with α-MEM medium (37oC, 5% CO 2 ) until they became confluent, then they were exposed to hydrogen peroxide for 4 hours. The protein content in the culture medium was measured by using Bradford protein assay at 655 nm wavelength. The result showed that hydrogen peroxide decreased protein concentration in the medium culture compared with group without hydrogen peroxide. Treatment group with chitosan concentration 0.4% and 1.6% exhibited a significant increasing of protein concentration in osteoblast culture medium compared with control. In conclusion, in osteoblast culture medium chitosan can inhibit the decreasing of total protein concentration which was caused by oxidative stress. Key words: osteoblast, hydrogen peroxide, chitosan, protein concentration

Amaranth-Protein Interaction in Food System and its Impact on Tryptic Digestibility

Abstract: Summary: Amaranth, a food color, is used in variety of food products to attract consumers, especially children. The purpose of the present study was to identify the component present in the food system which acts as a carrier of color and its distribution. The protein is the most possible candidate for color-conjugates and this was first explored by staining the resolved food proteins on PAGE simultaneously and separately with Amaranth as well as by Coomassie brilliant blue R250. It is the most widely used dye for protein assay. The color intensity of the Amaranth-protein complexes was slightly less than those of Coomassie brilliant blue R250, although the bands stained by Amaranth were very sharp, clearly separated and distinct. The staining procedure followed for Amaranth was quick. The impact of tryptic digestibility on amaranth–protein complexes has illustrated that dye may safely be used without any adverse effect. The possible mo0de of conjugation between amino acid and azo-bond is also discussed. The possible moode of conjugation between amino acid and azo-bond is also discussed.

Physicochemical characterization and DPPH radical scavenging activity of polysaccharide fractions isolated from Bupleurum chinense

Abstract: Bupleurum chinense DC., customarily called “Bei chaihu” in China, is a well-known traditional Chinese medicine distributed mainly in the northern region of China [1]. B. chinense is an important “harmony” herb, which can balance different organs and energies within the body, and it is also used as a tonic herb because of its ability to strengthen the action of the digestive tract, improve liver and circulatory system function, and relieve liver tension. However, the physicochemical characterization and pharmaceutical effects of bioactive polysaccharides from B. chinense have not been extensively studied. Therefore, the present studies were carried out to isolate and purify the polysaccharide fractions from B. chinense and further investigate their basic physicochemical characterization and antioxidant capacity. In the present study, the yield of the crude water-soluble polysaccharide extracted from B. chinense was 12.4% of dried material. After the freeze–thaw process and deproteination by a combination of proteinase and the Sevag method, the crude polysaccharide sample (WBCP) was further purified by AKTA Explorer purification with ion exchange chromatography and gel filtration chromatography. First, WBCP was loaded onto a DEAE-cellulose column eluted with de-ionized water and a 0 1 M gradient of NaCl solution. The main fractions eluted by de-ionized water and NaCl solution were collected, lyophilized, and further fractionated on a Sepharose CL-6B column eluted with 0.15 M NaCl solution. Three main fractions (WBCPa, WBCPb, and WBCPc) were separated for further analysis of physicochemical properties and antioxidant activities. Their chemical and physical characteristics were determined by chemical methods, gas chromatography (GC), and high-performance gel-permeation chromatography (HPGPC). The results of total sugar, protein, and uronic acid contents, molecular weight, and monosaccharide compositions of the polysaccharide fractions are summarized in Table 1. All the polysaccharide fractions, WBCPa, WBCPb, and WBCPc, appeared as pale yellow powders. The results using the phenol-sulfuric acid colorimetric method showed that the polysaccharide fractions WBCPa and WBCPc had a higher total carbohydrate content (98.1% and 99.2%, respectively) than WBCPb (95.7%). They showed a negative response in the Bradford assay. In addition, no absorption at either 280 or 260 nm was detected by UV spectrophotometer, which indicated the absence of protein and nucleic acid. Furthermore, based on the m-hydroxydiphenyl method, the uronic acid content of WBCPc was 33.5, while uronic acid was undetectable in both WBCPa and WBCPb. The average molecular weights of WBCPa, WBCPb, and WBCPc calculated by HPGPC were 103.7, 54.6, and 77.4 kDa, respectively. According to GC analysis, both of the neutral fractions, WBCPa and WBCPb, were composed of mannose, galactose, and glucose with molar ratios of 0.6:2.4:3.2 and 0.2:1.6:4.8, respectively, while the acidic fraction, WBCPc, was composed of mannose, glucose, and galacturonic acid with a molar ratio of 2.5:1:2.2.

Analysis of Proteins Bound to Contact Lenses

Abstract: The purpose of this study was to determine whether there are any proteins that attach to contact lenses that can be removed by cleaning solutions and if so, whether it is possible to quantify them by Bradford assay and identify them by nanoliquid chromatographytandem mass spectrometry (nanoLC-MS/MS). Our results indicate that the contact lenses indeed contain equal amounts of proteins, as determined by quantitative Bradford assay. NanoLC-MS/MS analysis led to the identification of several proteins, including lipocalin-1, lysozyme and tryptophan 5-hydroxylase. The significance of our findings is discussed.

Techniques to quantify the size of protein colloids in amyloid fiber formation

Abstract: A new method for the analysis of protein colloidal diameter has been developed using three existing protein concentration quantification techniques, absorption at 280 nm, colloidal gold assay, and DC protein assay. Protein colloids are formed in the process of aggregation and are thought to be intermediates in protein self-assembly and formation of amyloid fiber. Deposition of the protein fibers in tissues leads to numerous human diseases including Alzheimer’s. Lysozyme was incubated at pH 2.0, 55°C, an environment conducive to amyloid fiber formation. The protein colloids present in the supernatant of the samples after centrifugation were studied over a time course of 30 days. The OD 280 assay detects total protein concentration based on absorption of radiation in the near UV. The colloidal gold assay and DC protein assay only measure colloidal sphere surface protein concentration. Due to the surface plasmon resonance, the light absorption spectrum changes when proteins bind to colloidal gold particles. Using the measured protein concentration on the surface of protein colloids along with the total measured protein concentration in the entire protein colloidal spheres, an interior protein concentration for all colloids is obtained. The protein colloidal sphere size can be calculated by using the ratio between the interior protein concentration and total protein concentration. Results indicate that the colloidal gold assay, DC protein assay, and OD 280 assay can be used to quantify the size of the protein colloids. The colloidal gold assay and DC protein assay are both independently effective in analysis of surface protein concentration in protein colloids. The DC protein assay was found to be much quicker in data production as it only requires 15 minutes of incubation time. The DC protein assay was also more reliable than the colloidal gold assay in accuracy and precision of results.

The Comparison of Different Methods on Extracting and Measuring the Total Protein of Biofilms Formed by Enterococcus faecalis

Abstract: Objective: In order to measure the total protein of bacterial biofilm formed by Enterococcus faecalis ATCC33186,ultrasonication was used to extract the protein.After optimizing ultrasonic extraction conditions by different parameters,three methods commonly used to determine the protein concentration were taken.And we tried to confirm an accurate,rapid and simply method for quantifying protein in the biofilm.Methods: Ultrasonication was used to extract the protein from biofilm which was washed by PBS buffer after culturing 24 h.In order to determine the optimum ultrasonication condition,we chose different parameters as times,amplitudes,ultrasonic and interval times.After choosing the best ultrasonic treatment combined with SDS-PAGE electrophoresis,BCA,Bradford and Lowry methods were used to determine the total protein of biofilms.And we compared the data discrepancy of different methods.Results: The optimum condition of ultrasonication was ultrasonic time 2 min,Amp 20% and ultrasonic treatment for 2 s with interval of 2 s.The average concentration of total protein concentration of three times was 2299.1μg/dish by BCA,3793.8 μg/dish by Bradford,1858.0 μg/dish by Lowry.Conclusion: The optimum ultrasonic condition for protein extraction from Enterococcus faecalis biofilm was determined.The Bradford method was feasible and precise to determine the total protein concentration of bacterial biofilm.

The amino acid profile of yeasts from ketchup factory waste as a candidate of single cell protein (SCP).

Abstract: Yeast can be used as a feed additive to increase livestock health in the form of probiotic and immunostimulant. This research aims to observe the protein content and amino acid profile of yeast isolated from ketchup factory waste in East Java, Indonesia. The yeast biomass production was carried out in yeast malt broth medium containing 3% glucose, 0.3% malt extract, 0.5% bacto peptone and 0.3% yeast extract. Protein content in each yeast isolate was measured using Bradford method and the amino acid profile was estimated using High Performance Liquid Chromatography (HPLC). From seven yeast isolates detection resulted the highest protein content was KYP3K2 isolate containing 1172.33 µg/ml of protein. Besides, both isolates KYP3K2 and AYP6K2 have low nucleic acid content of 15.8 %. Amino acid profile showed that both samples consist of nine essentials amino acid and seven non essentials amino acid. In conclution, both samples fully recommended as Single Cell Protein as feed livestock.

Identification of Proteins in Tissue Fluids of the sea mussel Isognomonalatus

Abstract: The aim of this preliminary study was to investigate the presence of proteins in the tissue fluids of the sea mussel Isognomonalatus. Protein extraction was done by the chloroform-cold ethanol technique. Immunization for production of antibody to be used as reagents in Western blotting, assessment of the protein concentration by the Bradford method, protein characterization by native polyacrylamide gel electrophoresis (PAGE) were also performed as a part of the methodology of this study. The results showed a protein content of 65 mg/ml in tissue fluids and a protein band approximately of 220 kDa in PAGE that was further confirmed by the Western blotting. Future work should investigate the structure and function of the proteins separated from the tissue fluids and we considered it as a limitation of this investigation. The sea bivalve literature is scanty. However the limitation of this work we still can conclude that there are high molecular weight proteins in large concentrations in tissue fluids of the sea mussel Isognomomalatus.

Comparison of four cytotoxicity assays for determining the acute toxicity of diesel exhaust particulate extracts

Abstract: OBJECTIVE To compare the determination ability of four in vitro cytotoxicity assays for acute toxicity of diesel exhaust particulate (DEP) extracts. METHOD The cell viability of cultured 16HBE cells was measured by the MTT assay, the neutral red assay, the lactate dehydrogenase (LDH) leakage assay and the determination of total protein content after 12, 24 and 48h of being exposed to DEP extracts at different concentrations of 5, 10, 20, 40, 80 and 160 microg/ml. RESULTS For measuring the acute cytotoxicity, the MTT assay was more delicate than the other three methods after 12h exposure. The sensitivity of both the MTT assay and the neutral red assay increased as the exposure time extended. The cell viability at each dosage decreased significantly after being exposed to DEP extracts for 24h compared to the control group (P < 0.05). When using the LDH assay, the cell viability reduced after 24h exposure to DEP extracts, but changed no more as time prolonged. Only the cell viabilities of the groups exposed to the high dosages of DEP extracts were observed with significant reduction by the protein assay after 12h and 24h exposure respectively. The cell viabilities measured by the same method at all dosages decreased after 48h exposure. CONCLUSION The results of the four cytotoxicity assays showed differences in the present study. The MTT assay and the neutral red assay are more sensitive in detecting the adverse effect to 16HBE cells done by DEP extracts compared to the LDH assay and the protein assay. Taken the indicants of each toxicity assay into account, the DEP extracts may have major adverse effects on the functions of the organelles in 16HBE cells, such as mitochondria and lysosomes, however little influence to the cell anchorage dependence and the cell membrane damage.

Concentração de proteína solúvel por bradford revela diferenças no metabolismo de plantas de ora-pro-nobis em diferentes doses de nitrogênio

Abstract: Ora-pro-nobis (Pereskia aculeata Mill.) is a protein-enriched food, widely used in rural communities, presenting increasing importance in the food and pharmaceutics industries. The aim of this study was to determine the protein concentration by the Bradford and Kjeldahl methods in plant leaves of ora-pro-nobis under different nitrogen fertilizer levels, comparing these methods. Leaves of ora-pro-nobis from plants fertilized with different nitrogen levels (0, 50, 100, 200, and 400 kg N/ha) were harvested at 423 days after planting (DAP). For the method of Bradford, the leaves were grounded in liquid nitrogen and macerated in 50 mM Tris-HCl, pH 7.0, the homogenate was centrifuged and the soluble proteins were determined in the supernatant. To evaluate the protein profile, samples of the different treatments were separated by 14% SDS-Tricine-PAGE. The traditional method of Kjeldahl was performed using the correction factor 6.25. For both methods, the results indicated that there were changes in the concentration and composition of proteins present for different availability of N in the soil. The total protein by Kjeldahl increased up to the level of 100 kg N/ha, and the soluble protein by Bradford increased in N levels between 50 and 200 kg/ha. By SDS-Tricine-PAGE, there was an increase in intensity of bands in line with the Bradford method results. These results suggest that the evaluation of the soluble protein by the Bradford method allows assessing differences in the metabolism of ora-pro-nobis plants, expressing biological information that was relevant to physiological and nutritional studies.

Protein assay apparatus

Abstract: A protein assay apparatus includes at least one light-emitting unit for generating a first light, a second light or a third light for BCA protein assay, Bradford protein assay or Lowry protein assay, and at least one light-sensing unit for sensing the absorbed light passed through the test sample to calculate the concentration of protein in the test sample. This design shortens the protein examination time and greatly reduces the protein examination cost.

The Reactivity and Allergenic Potential of Hazelnut Peptides

Abstract: The aim of this paper was to focus on proteins present in some food products, like hazelnuts and to investigate their allergenic potential. Several techniques were used to characterize these extracted proteins, with respect to their composition, degradability by digestive proteolytic enzymes and their reactivity with specific antibodies. It was important to analyse which proteins were present in the hazelnuts, to see if there were proteins present to trigger an allergic reaction and if the digestion enzymes trypsin and pepsin influence the presence of the (allergic) protein compounds. Allergies to tree nuts and seeds can cause life-threatening and sometimes fatal reactions. To examine the properties of Hazelnut protein it was important to solubilize it by extraction. After extraction, it was investigated how hazelnut protein can be modified by proteases and what the effect was on the immune reaction. The Bradford method is a fast and sensitive method to determine the concentration of soluble protein. When the Bradford reagent (Coomassie Brilliant Blue) binds to the protein, the colour changes from red to purple and the absorption maximum changes from 495 to 595 nm. The value obtained as the final concentration of proteins was 7.3495. SDS-PAGE is a method to separate mixtures of proteins by electrophoresis. Protein molecules are negatively charged by binding of SDS molecules; subsequently they are separated in an electric field. Their differences in size (molecular weight) leads to separation. In this case the method is used to follow proteolytic degradation of hazelnut proteins (allergens) by intestinal proteases (trypsin, pepsin). A different, more specific and sensitive method is immunoblotting (Western Blot) in which the SDS-PAGE separated proteins are transferred from the gel to a membrane and specific antibodies are used in a series of reactions to visualize specific allergens on this membrane. The remarked spots represented a positive identification of allergenic proteins. This means that peptide fragments of various size, produced during the digestion of a protein can still be immunological active. As it was shown there was still reactivity between proteins and specific antibodies. The Dot Blot is a simple immunoblotting technique used to detected specific proteins in a mixture of different proteins and/or other molecules. No separation technique prior to the actual immuno-detection is necessary. Also, Dot Blot confirmed the presence of allergenic proteins made visible through the light spots on the membrane.