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. 

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. 

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

Bradford protein assay, see Coomassie dye binding assays... 

Bradford Protein Assay Kit (Pierce, Rockford, IL), including protein standards (Pierce)... 

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...

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. 

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. 

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]. 

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). 

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