Bradford method

The Lowry and Bradford methods have been compared for protein quantitation in samples containing membrane fractions.7 While the Lowry method yielded reproducible results over 2 weeks of sample storage at 20 °C, the Bradford method was shown to significantly underestimate membrane proteins, even after 

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Protein concentration was determined by the Bradford method (16) using bovine serum albumin as a standard. 

The WCE is clarified by two successive centrifugations at 16,100 for 2 and 10 min at 4°, respectively, using an Eppendorf F 45-24-11 rotor in a table centrifuge. After each centrifugation, the supernatant is carefully transferred to a new precooled Eppendorf tube, avoiding the lipid layer, and the total protein concentration (mg/ml) is estimated using the Bradford method (Biorad). 

Protein content The amount of protein in each extract can be determined by the Bradford method (Bradford, 1976), using BSA as a standard. Briefly, make a standard curve with 0,2,4,6,8,10,15 and 20 pg / mL BSA and mixed with 1 mL of Bio-Rad protein assay (diluted 1 4). Read standard curve and samples at A595 in a spectrophotometer, using as blank 1 mL of diluted Bio-Rad protein assay. 

Dowers Grove, IL), insulin by a radio immunoassay by means of Coat-A-Count Insulin Detection Kit (Diagnostic Products Corp., Los Angeles, CA), and proteins by Bio-Rad Protein Assay (Bradford) method (Bio-Rad, Hercules, CA). 

Bradford method Bicinchonic acid method Peterson method... 

The protein content in HNL was measured by the Bradford method using a Bio-Rad protein assay kit and bovine serum albumin as the standard." The protein content of crude HNL after the fractionation with 30 % (NH4)2S04 saturation was found to be roughly 10 mg mL and the activity was 120 U mL (specific activity 12 U mg ). 

A detailed discussion of Folin-Ciocalteu s phenol protein determination method, especially with respect to possible disturbances and troubles and in comparison with the Bradford method, is given by Peterson (1996) loc. cit. 

Water extract of W. sativa was prepared, dried and powdered. Powder was dissolved in phosphate buffer saline (pH 6.4) and centrifuged at 10.000 rpm for 30 min at 4 °C. The supernatant was collected as the soluble extract by removing the oily layer and unsoluble pellet. Protein concentration of the soluble extract was determined with Bradford method. Then proteins dialyzed against 0.05 M phosphate buffer (pH 6.4) using 3500 MW cut off dialyzing bags and centrifuged. 

Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur. Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute. Table 1.1 contains such a series of count rates obtained with a scintillation counter on a single radioactive sample. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample (the counting rates, the velocity of the reaction, or protein concentration) and dividing the total by the number of times the measurement was made. The mean is defined by Equation 1.1. 

Example 2 Ten identical protein samples were analyzed by the Bradford method for protein analysis. The following values for protein concentration were obtained. 

The reaction that leads to BCA color formation as a result of the reduction of Cu2+ is also strongly influenced by the presence of any of four amino acid residues (tyrosine, tryptophan, cysteine, or cystine) in the amino acid sequence of the protein. Unlike the Coomassie dye-binding (Bradford) methods, which require a minimum mass of protein to be present for the dye to bi nd, the presence of only a single amino acid residue in the sample may result in the formation of a colored BC A-Cu+ chelate. This is true for any of the four amino acids cited above. Studies done with di- and tripeptides indicate that the total amount of color produced is greater than can be accounted for by the simple addition of the color produced with each BCA-reactive amino acid, so the peptide backbone must contribute to the reduction of copper as well. 

Determine the total protein in triplicate by the Bradford method using bovine serum albumin as standard solution [2], Determine the enzymatic activity of jack fruit crude extract in triplicate by measuring the absorbance at 410 nm of o-quinone produced by the reaction between 2.8 mL of 0.05 mol L 1 catechol solution and 0.2 mL of supernatant solution in 0.1 mol L 1 phosphate buffer solution (pH 7.0) at 25°C. The initial rate of enzyme-catalyzed reaction is a linear function of time for 1.5-2.0min. One activity unit is defined as a quantity of enzyme that causes the increase of 0.001 absorbance per minute under conditions described above [1]. 

N.J. Kruger, The Bradford method for protein quantification. In J.M. Walker (Ed.), The Protein Protocols Handbook, Humana Press, New Jersey, 2002, pp. 15-21. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

In order to calculate enzyme activity, one should divide PNP concentration obtained per minute by the enzyme concentration in the test tube. In order to determine the enzyme concentration in the test tubes, we will apply the Bradford method (Bradford, 1976). 

Kruger, N.J. (1994) The Bradford Method for Protein Quantitation, Methods Mol. Biol. 32, 9-15. 

CFFs (0.15 ml each) were serially collected from the pulmonary artery prior to ischemia and during reperfusion. Fractions were collected in the reperfiision phase (10 first CFF samples and 5 CFF samples — after 2, 5, 10, 15 and 20 min of reperfiision, respectively). Iron and copper concentrations in the CFFs were measured according to standard procedures, using a Zeeman atomic absorption spectrometer (Varian, Spectron AA-300/400) [30]. Protein content was determined according to the Bradford method [31]. 

The fractions recovered from the column are assayed for purity by SDS-PAGE (the monomeric toxin of 33kDa is inevitably accompanied by heptamers which are detected in the gel if the samples are not boiled). The protein concentration is determined by measuring A280 (which is 0.19 for 0.1 mg toxin/ml) or by the Bradford method (A595 = 0.6 for 10 [xg toxin/ml assay mixture). 

Under these conditions, the product exhibits a broad absorption band between 560 and 580 nm, and a microtiter plate reader set at 575 nm may be used for measurement. Correlation of absorbance with protein concentration has been performed using several protein standards, as shown in Figure 1.4, which also shows results obtained with the same protein standards by the Bradford method. These data demonstrate much better sensitivity with the ninhydrin method, and suggest an 10-fold improvement in detection limit. An important advantage of this method is that the differences in calibration curves obtained using different protein standards are relatively small. Interferences include free amino acids as well as other compounds containing amine groups. 

Figure 1.4. Comparison of results of ninhydrin and Bradford methods for total protein concentration in tissue samples. Proteins in both assays are the same.8 [Reprinted, with permission, from B. Starcher, Anal. Hiochem. 292, 2001, 125-129. A Nirhydrin-Based Assay to Quantitate the Total Protein Content of Tissue Samples. Copyright 2001 by Academic Press.]...

Assays. Samples collected in all experiments were cooled and stored at 4°C, then the concentration of total protein in the solution was assayed by the Bradford method (22), and the concentration of p-lactamase was assayed according to Sykes and Nordstrom (23). 

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