Determination Bradford assay

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

Protein concentration was determined using the Bradford assay at 595 nm. 100 pL of the sample were introduced into a cuvette containing 5 mL of Bradford solution (100 mg of Coomassie blue, 50 mL of ethanol and 100 mL of 85 % phosphoric acid dissolved in 850 mL of H2O). The solutions were incubated for 5 min at room temperature. The absorbance was measured at 595 nm. The protein concentration in the sample was determined using a calibration curve plotted with serum albumin (1 mg mL ) as a standard.)... 

Protein concentrations were determined by performing Bradford assay using bovine serum albumin as a standard. Equal amounts... 

THE COOMASSIE DYE-BINDING (BRADFORD) ASSAY FOR DETERMINING TOTAL PROTEIN... 

Determine the protein concentration of the soluble protein extracts by Bradford assay (20) to confirm that effective cell lysis has occurred see Note 5). 

We generally assess the protein concentration of new chemokine stock solutions by OD280 using the appropriate extinction coefficient for each chemokine or by Bradford assay. Although this is not often practical when very small quantities of chemokine are purchased because 10-20% of the material may be sacrificed for an accurate determination, we have found that the actual protein concentration may vary by as much as 2CM-0% between lots which can significantly effect the reproducibility of chemotaxis assays. 

Dye-Binding (Bradford) Assay. The binding of proteins to Coomassie Brilliant Blue 250 causes a shift in the absorbance maximum of the dye from 465 nm to an intense band at 595 nm. Determination of the increase in absorbance at 595 nm as a function of protein added provides a sensitive assay... 

Tris buffers Tris is also a much used buffer. However, it has one great disadvantage its pH is highly dependent on temperature and concentration. The pH of a Tris buffer will increase from 8.0 at 25 °C to 8.6 on cooling to 5 °C and on dilution of a 0.1 M solution at pH 8.0 to 0.01 M, the pH will fall to 7.9. This problem can only really be avoided by adjusting the pH of the buffer under the conditions of temperature and concentration where it is to be used. In addition, Tris has been shown, like phosphate discussed above, to interfere with many enzymic reactions, particularly those which have aldehyde intermediates. It also interferes with many chemical reactions, like the coupling of proteins to activated surfaces, and the Bradford assay for spectrophotometric determination of proteins. 

The enzyme activity was measured by a continuous spectrophotometric assay (see Methods), active site concentration was determined by FAD absorption at 452 nm (8 = 12.83 mM-icm-i) as described by Frederick et al. (1990) and the protein concentration was measured by the Bradford assay (BioRad reagent) using bovine serum albumin as standard, or by its absorption at 280 nm using a published factor of 1.67 O.D. per mg (Swoboda Massey, 1965). The specific activity was 430 U/mg, and the overdl yield of enzyme, based on active sites measurement (452 nm absorption), was about 40%. 

The importance of a reliable assay for the target protein cannot be overemphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein. 

The protein concentration in each fraction is determined by using Bradford assay. 

Use lysis buffer if needed to adjust for equal loading among the samples according to the protein concentrations determined in the Bradford assay. 

The protein content of the commercial enzyme preparations and the fungal supernatant samples were determined using the Bradford assay [23]. 

To determine the protein concentration and purity, check a sample of each fraction in a Bradford assay (or in a photometer at 280 nm absorption) and by SDS-PAGE followed by Coomassie blue staining. 

Furthermore, every protein determination is sensitive to detergents or certain ions. Hence, when presenting a concentration value it is good practice to also mention the assay that was used as well as the benchmark protein. The methods of choice are the Bradford assay and the BCA (bicinchoninine acid) assay. Anyone working with membrane proteins and detergents should use the BCA assay. Otherwise, the choice between the BCA and the Bradford assays seems to be a question of taste. 

When performing preparative small-scale assays (4 mL polypropylene columns), total protein concentration in the collected fractions is determined by using a standard Bradford assay protocol with Coomassie Plus protein assay reagent. The assay is performed in 96-well standard microtiter plates. 

Using a Bradford assay, determine the protein concentration of the P2 fraction, which was previously resuspended in isolation buffer. To do this, mix 799 //I water with 1 //I P2 and add 200 /il Bio-Rad Protein Assay reagent (Hercules, CA). Incubate for 5 min at room temperature and determine the absorbance at 595 nm. To convert the absorbance value to protein concentration, make a calibration curve with a series of known concentrations of bovine serum albumin. Adjust the concentration of the P2 suspension to obtain 1 mg protein/100 /il solution. 

Determine the protein concentrations of the extracts using the Bradford assay. 

Determine protein concentration using the Bradford protein assay. If the Bradford assay is not used, make sure that the presence of glutathione in the sample does not affect the colorimetric assay being used. Run a sample of the eluted protein on an SDS-PAGE gel to confirm the protein is of the expected size and is not degraded. 

Note Twenty-four liters of culture produced approximately 9 ml of GAL4VP16 protein with a protein concentration of 1.35 mg/ml as determined by Bradford assay. The final preparation should be greater than 95% pure as judged by SDS-PAGE. 

Thaw one aliquot of dissolved thioester [Ala ]-SDF-1(1-49)-MESNA (Subheading 3.3) on ice and determine protein concentration by spectrometric measurement (dilution in BB, 28o=1,490/M Vcm )- Therefore dilute a sample immediately before measurementin order to preventaggregation. Alternatively, determine protein amount by performing a Bradford assay. Concentration ranges between 5 and 10 mg/mL. 

The protein content was determined using a commercial assay kit (Bio-Rad Protein Assay kit) with Bovine Serum Albumin as standard, following the procedure described by Bradford [13]. 

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

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. 

The Bradford protein assay as described in Chapter 2 is based on the absorbance change that occurs upon binding of Coomassie Blue dye to proteins. Explain how you would study the dynamics of this binding process and experimentally determine the number of binding sites on a protein. 

Several useful methods for the quantitative determination of protein solutions were discussed in Chapter 2. Two of those methods, the Bradford protein assay and the direct spectrophotometric assay, will be applied to the a-lactalbumin solutions. Neither of these assays is specific for a certain type of protein rather they both estimate total protein content. 

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