The Bradford protein assay

Protein concentration of the sample is quantitated by the Bradford protein assay with bovine serum albumin used as standard. 

Example 4 The Bradford protein assay is one of the most used spec-trophotometric assays in biochemistry. (For a discussion of the Bradford assay, see Chapter 2.) Solutions of varying amounts of a standard protein are mixed with reagents that cause the development of a color. The amount of color produced depends on the amount of protein present. The absorbance at 595 nm of each reaction mixture is plotted against the known protein concentration. A protein sample of unknown concentration is treated with the Bradford reagents and the color is allowed to develop. 

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. 

Compton, S.J. and Jones, C.G. 1985. Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 151 369-374. 

A 40-pL sample of the supernatant is removed for total protein analysis. The Bradford protein assay (14) is suitable, because it is compatible with the lysis buffer. 

The protein concentration in solution was measured by the Bradford protein assay using BSA as a standard (Bio-Rad). The Bradford colorimetric method cannot distinguish the BSA protein from enzyme protein. Therefore, no differentiation was possible between BSA and enzymes in solution other than by measuring the enzyme activity. The enzyme activities in solution including FPU and CBU were measured according to the methods described by Ghose [35]. 

Protein Determination. Protein was monitored by reading the absorbance at 280nm or by using the Bradford protein 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. 

All fractions were analysed for AE activity and protein content. The protein content was measured spectrophotometrically according to Bradford using the BioRad protein assay kit with Y-globulin as standard (5). 

In 1976, Marion Bradford introduced the first Coomassie dye-based reagent for the rapid colorimetric detection and quantitation of total protein. The Coomassie dye (Bradford) protein assay reagents have the advantage of being compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples. 

Light measurements are recorded by the luminometer software GloMax and protein concentrations in the supernatants are measured using a Bradford Protein Assay Kit following the manufacturer s protocols and analyzed with an EM680 microplate reader. 

Table 1 Comparison of the recombinant production and affinity chromatographic purification of GFP from B. megaterium [30]. Different affinity tag fusion forms of GFP were produced in II. megaterium WH323. Purification was performed using affinity chromatography. Amounts of purified GFP-Strep were determined using a Bradford protein assay kit (Bio-Rad Munich Germany) and BSA (Perbio Rockford USA) as standard. Amounts of purified GFP-His, His-TEV-GFP, Strep-Xa-GFP and Strep-TEV-GFP were calculated via their relative fluorescence per mg protein...

To compare activity between samples, all are normalized for total cytosolic protein. We carry this out using the BioRad protein assay (a commercial preparation of the Bradford assay), and a standard curve from BSA protein standard... 

Determine the total protein content of the supernatant (crude extract) by performing a Bradford protein assay. 

Samples were assayed by absorbance at 280 nm, Bradford protein assay(.27), and radial immunodiffusion plates (Helena Laboratories). Alpha-1 antitrypsin was assayed for biological activity by competitive assay with elastase (28). DNA was assayed by the diphenylamlne methods of Burton(29) and Giles and Mvers(30). with modifications due to the presence of PEG and salts (31.). Materials were also assayed by size exclusion chromatography on Superose 6 (Pharmacia FPLC), with peak integration at 280 nm. PEG was assayed by HPLC. 

Bradford protein assay stock solution dissolve 150 mg of Coomassie Brilliant blue-G250 in 50 mL of ethanol (96%, HPLC grade). Mix extensively using a magnetic stirrer Filter the stock solution using Whatman No 1 filter paper and store the filtrate in the dark at 4°C. This solution is stable for several months (see Note 1). 

Bradford protein assay working reagent Add 3 mL of Bradford stock solution to 97 mL of 7% phosphoric acid in water. Prepare the working reagent at least 1 d in advance of use. Bradford working reagent is stable for 1 mo when stored in the dark and at room temperature (see Note 1). 

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. 

Fig. 7. Coomassie Brilliant Blue R-250 (I) is used to stain proteins, e.g., after gel-electrophoretic separation, its derivative G-250 (II) is applied in the Bradford assay for protein quantification... 

The first assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein. Amb a 1 is approximately 1% of the total protein extracted from ragweed pollen hence the Bradford assay does not reflect Amb a 1 concentration. However, at this step of the production process, the protein concentration is used to calculate final yields and not to make time-dependent or expensive decisions. Hence the nonspecific Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. 

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.)... 

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 ). 

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