Protein Bradford

Bovine serum albumin (BSA) is often used as a calibration standard, but it has greater general dye-binding capacity than most proteins (Bradford, 1976). 

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

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). 

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

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]. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

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). 

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. 

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). 

The concentration of protein in the lysate should be determined as a guide to even loading of gels or the amount of material to be subjected, for example, to immunoprecipitation. A simple and reliable method for this is that of Bradford (Bradford, 1976). 

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 reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

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... 

Nsahlai IV, Green H, Bradford M, Bonsi MLK (2002) The influence of source and level of protein, and implantation with zeranol on sheep growth. Livestock Prod Sci 74 103-112 Parry DW, Jenkinson P, McLeod L (1995) Fusarium ear blight (scab) in small-grain cereals - a review. Plant Pathol 44 207-238... 

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 content Bradford s test was performed to determine the protein content (mg g ) of the lyophilisates. A sample of the lyophilisate (2.5 mg) was dissolved in glycylglycine (Gly-Gly) buffer (1.5 mL). An ahquot of this solution (20 gL) was diluted with glycylglycine (Gly-Gly) buffer (30 gL) and Bradford s solution (950 gL) was added. After 5 min, the absorbance was measured at 595 nm. The concentration of FSA is calculated from the interpolation of the calibration curve of bovine serum albumin (BSA) 437 mg total protein/gram of lyophihsate. 

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 was determined according to Bradford (1976) using ovoalbumin 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 ). 

The protein content is measured by biochemical methods, such as methods of Lowry or Bradford [14,18,19,21,23], now usually by commercially available kits. The DNA content can be measured by flow cytometry, and the chromosomes can be counted directly in cells after their arrest in the metaphase of the mitosis by Colcemide [21]. 

Fig. 7.16 Saturation isotherm of NO 711 binding to mGATl-membrane fraction as measured in MS binding experiments. One representative example from a series of identical experiments is shown. Total binding of NO 711 ( lOpg protein according to Bradford). Nonspecific binding (o) measured as binding of NO 711 in the presence of 10 mM GABA. Each data point depicts the mean + SEM from triplicate values.

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