Showing papers on "Bradford protein assay published in 2010"

Linearization of the Bradford Protein Assay

Abstract: Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

Highly sensitive protein concentration assay over a wide range via surface-enhanced Raman scattering of Coomassie brilliant blue.

Abstract: In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilliant blue G-250 (CBBG) to proteins. In a protein-CBBG liquid mixture, surface-enhanced Raman scattering (SERS) is sensitive to the amount of unbound CBBG molecules adsorbed on silver surfaces, and the bound CBBG amount is directly related to the target protein concentration. Accordingly, a novel method for detecting total protein concentration in a solution has been developed based on SERS of unbound CBBG with an internal standard of silicon. Two obvious advantages of the proposed protein assay over conventional Bradford protein assay are its much wider linear concentration range (10(-5)-10(-9) g/mL) and 200 times lower limit of detection (1 ng/mL), which demonstrates its great potential in rapid, highly sensitive concentration determination of high and low-abundance proteins.

Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

Abstract: Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

Abstract: Background: The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. Aims: We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. Material & Methods: Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. Results: We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. Conclusions: We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.

Moving Enzyme-Linked ImmunoSorbent Assay to the Point-of-Care Dry- Reagent Strip Biosensors

Abstract: In this work, we described a point-of-care (POC) dry-reagent strip biosensor (DRSB) based on enzyme tracers and portable strip reader for simple, low-cost and sensitive assay of protein detection in minutes. Horseradish Peroxidase (HRP) and Rabbit IgG (R-IgG) were used as a model system for the demonstration of the proof-of-concept. The sandwich-type immunoreactions were performed on the DRSB and the HRP tracers were captured on the test zone of the biosensor. The excess of HRP tracers were captured on the control zone of the biosensor through the immobilized secondary antibody. Subsequent enzymatic reaction in the presence of the substrate produced insoluble enzymatic products, which deposited on both test and control zones of the DRSB and formed two characteristics blue bands. While qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the intensities of the test zone with a portable “strip reader”. The quantitative response of the optimized DRSB over the range of 0.1-50 ng mL -1 IgG in association with a 10-min assay time is obtained, and the limit of detection is estimated to be 0.05 ng/mL, which is ten times lower than that of the gold nanoparticle (GNP)-based DRSB. The enzyme-based DRSB was used to detect Carcinoembryonic Antigen (CEA) biomarker in human plasma successfully. Such enzyme-based DRSB offers a simple and fast tool for point-of-care protein assay and a potential substituent for the traditional Enzyme-linked Immunosorbent Assay (ELISA).

Development and validation of an improved Bradford method for determination of insulin from chitosan nanoparticulate systems.

Abstract: Context: Blank chitosan nanoparticles are currently used as reference for the calibration curve, which fails to resolve the supernatant of the nanoparticles in the interference of Coomassie Brilliant Blue G-250 reagent; supernatants are generated at different chitosan nanoparticulate prescriptions, which have different interferences. There are notable errors in the experimental results, and the method is not feasible.Objective: In this study, an improved, rapid, and economic Bradford method was developed and validated.Materials and methods: The pH of the supernatant of blank chitosan nanoparticles was adjusted to 7–9 through adding saturated NaOH. The precipitation (free chitosan) in the solution was separated by centrifuging for about 10 min (4000 r/min).Results: The method eliminated the interference of free chitosan of different prescriptions. The results showed that the method presented a linearity in the range of 50–300 μg/mL (R2 = 0.9992), and possessed a good inter-day and intra-day precision based...

Double antibody complex retinol-binding protein assay kit

Abstract: A retinol-binding protein assay kit in the market at present has good specificity and insufficient sensitivity or has high sensitivity and poor specificity because the purity of antibodies is insufficient or the potency cannot meet the requirement. The invention provides a double antibody complex retinol-binding protein assay kit, which consists of three parts, namely, a reagent R1, a reagent R2 and calibration materials. The reagent R1 is a phosphate buffer system which consists of phosphate buffer solution with the pH of 7.2 to 7.6, polyethylene glycol 6000-8000 and ethylene diamine tetraacetic acid; the reagent R2 is antibody solution which consists of mouse anti-human monoclonal antibody, rabbit anti-human polyclonal antibody, phosphate buffer solution with the pH of 7.2 to 7.6 and the ethylene diamine tetraacetic acid. By adopting a complex antibody of the polyclonal antibody and monoclonal antibody, the sensitivity and high linearity are guaranteed, and the accuracy of the measured result is greatly improved.

Determining protein patterns for three fungus species Aspergillus fumigatus, Asp. flavus and Asp. niger, obtained from outdoor air in Iran.

Abstract: Aspergillus species are saprophytic fungi widely distributed in nature and are associated with a number of human disease. The aim of this study was to compare electrophoretic protein patterns Aspergillus fumigatus, Asp. flavus and Asp.niger and identify the differences of three protein patterns. In this study, three species of Aspergillus fumigatus, Asp.flavus and Asp.niger which were separated from outdoor air in Iran were used to compare electrophoretic protein patterns antigens isolated of Aspergillus. First, these fungi were grown in saboraud glucose agar and preserved at 27°C for 48-72 hours and then they were cultured in saboraud glucose broth to provide protein extract of above mentioned fungi and Bradford method was used in order to measure the level of fungi extracts protein. Protein was dissociated by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 11% separating gel. The gel was stained with coomassie brilliant blue G250 and after stabilizing, gel staining and disstaining, different protein bands were appeared. Results revealed, 69 protein bands with molecular weights between 11/5 and 178KD. Among these bands, protein bands with molecular weights of 15, 23/5, 27, 33/5 and 61KD are in two species of Asp.fumigatus and Asp.flavus, protein bands 28/5, 40 and 47KD are in two species of Asp.flavus and Asp.niger, the band with 120 KD is in Asp.fumigatus and Asp.niger and protein bands 23, 35 and 36 KD are among these bands which were presented in every three species of Aspergillus. In addition, a number of strong and weak protein bands were recognized. It was concluded that there was no meaningful relation between protein bands, which were obtained from isolated fungal sp. under study and these three isolates did not follow the same electrophoretic protein patterns.

The effectiveness of soluble protein extractability under the effect of pH, molarity and type of buffers of three different major skeletal muscles in cattle.

Abstract: The present study was conducted in an attempt to study the effects of the type, pH and molarity of extraction buffer on protein extractability from beef Longissimus dorsi (LD), Supraspinatus (SS) and Semitendinosus (ST) muscles. All muscle samples were dissected out from carcass immediately after slaughter and subjected to extractions using freshly prepared buffers of different buffer type (Tris Base and Tris HCl), pH (8.3 and 7.5) and molarity (100 and 20 mM). Following extraction, the total extractable protein concentration was determined by Bradford assay. The results exhibited significant (p<0.05) effects of muscle type X buffer interaction on extracted total protein concentration. The statistical analysis also revealed interaction between type of buffer, pH and molarity significantly (p<0.01), affected the extracted protein concentration. Based on the results, optimal buffer suggested to use for muscle protein extraction is Tris-Base with pH 8.3 and 100 mM. The present study demonstrated that the extractability of skeletal muscle protein was significantly influenced by the type, molarities and pH of the extraction buffers used.

Isolation of Plasma Proteins from Bovine Blood by Cold Ethanol Precipitation and Anion Exchange Chromatography

Abstract: Plasma (100.00 ml) obtained from bovine blood by centrifugation was fractionated into seven precipitates by cold ethanol precipitation. The yield (amount) of proteins in the precipitates calculated from the standard curve of BSA was 1160.83 mg, 806.57 mg, 1149.94 mg, 8.79 mg, 19.88 mg, 21.98 mg, and 13.97 mg respectively, while the total amount of protein obtained was 3180.00 mg. The precipitates were fractionated into 25 fractions each by Anion Exchange Chromatography (AEC) and the amount of protein in each fraction was obtained by Bradford protein assay. SDS-PAGE analysis was performed on the fractions with proteins and their estimated molecular weights were obtained with the aid of the molecular weight marker. The result indicated that cold ethanol precipitation and anion exchange chromatographic techniques are valuable tools for the purification of bovine blood to obtain high grade α, β and γ-fibrinogen, IgM (μ-globulin), IgG (γ -globulin), alpha (α –globulin), β-globulin (E-globulin) and albumin. Keywords: Plasma; Chromatography; Electrophoresis; Albumin; Fibrinogen; Globulin DOI: 10.3126/jncs.v26i0.3624 Journal of Nepal Chemical Society Vol. 26, 2010 Page: 2-12

Alginic acid-based macromolecular chemiluminescent probe for universal protein assay on a solid-phase membrane

Abstract: A novel chemiluminescent (CL) technique for the rapid determination of proteins on a membrane is described. The method utilizes an interaction between luminol-labeled alginic acid macromolecule and proteins. The synthesis of the macromolecular probe consists of the oxidation of alginic acid with NaIO4, the introduction of luminol through imine formation as a CL tag, and the reduction of the conjugate with NaBH4 to obtain the stable probe. The analytical protocol consists of adsorbing proteins on a poly(vinylidene difluoride) (PVDF) membrane, incubating the membrane for 30 min with the probe solution in the presence of boric acid and a surfactant, two short washing steps in order to remove an excess of the probe, and detection of CL intensity with hemin, tetra-n-propylammonium hydroxide and H2O2. This proposed CL assay for proteins can be finished within 1 h, and indicates the detection limit of 15–250 ng of proteins on the membrane. The CL signals in the calibration curves for some proteins such as albumin show proportional intensities against the amounts of the proteins less than ca. 125 ng, though there is a logarithmic relationship between the CL signals and the protein amounts larger than ca. 125 ng. However, some other proteins indicate the proportional CL intensities against the increasing amounts in wider range up to 500 ng of the proteins. The synthesised alginic acid-based probe indicates specific selectivity towards proteins, and should be used as a CL probe for the universal detection of various proteins on a solid-phase membrane even in the presence of DNA and RNA.

The Determination of the Protein Content in Food

Abstract: According to the current standard of Determination of Protein Content in Food GB/T5009.5-2003 in China,there are two methods to determine the protein content.They are Kjeldahl method of nitrogen determination and Dumas method.Both of the two ways determine the nitrogen content by nitrification firstly and then calculate the protein content according to the conversion coefficient between the nitrogen and protein.If such high nitrogen content chemical substance as Melamine is added to the food,the protein content will be higher accordingly.In this peper,four direct determination methods were introduced including Bradford method,Biuret method,Lowry method and ultraviolet absorption spectrometry.These methods are more precise to determine the protein content in food than Kjedahl method which often overstates the protein content due to adding the high nitrogen-containing substances.

Effect of ionic strength and pH on stability of heat-stable protein in the barley

Abstract: Heat-stable protein in the barley takes an important role in the non-biological stability and turbidity in the beer.The Bradford method and SDS-PAGE were used to study the stability of heat-stable protein in the barley after changing pH and ionic strength.The results showed that both pH and ionic strength took an important role in the stability of heat-stable protein,the influence of alkaline environment was far bigger than acid environment,ionic strength was inversely correlated with the stability of heat-stable protein.With the ionic valent states increasing,stability of heat-stable protein reduced.The study provided reliable parameters for the study on characteristics of heat-stable protein,and provided references for pertinently improvement in beer production.

Development of Protein Assay in Natural Rubber Latex by the Designed Spectropantonometer

Abstract: In this research, protein assay in natural rubber latex (NRL) was developed by using Lowry and Biuret methods for color developing followed by analysis with a laboratory designed and fabricated spectropantonometer. It was found that the developed method could directly be used to quantify colloidal protein in latex without precipitation and preconcentration. Therefore, this protein assay could directly be used to detect the intensity of the color developed proportional to the concentrations of protein in natural rubber latex. From the experiment, the proteins found in natural rubber are 0.037 and 0.044 %w/v by Lowry and Biuret methods, respectively. The results obtained from both color developing methods are not significantly different at 95 % confidence limit. Keywords: natural rubber latex, protein, Lowry method, Biuret method