Bradford Protein Determination

G. G3 Calibration curve. (You can do this exercise with your calculator, but it is more easily done by the spreadsheet in Figure 4-13). In the Bradford protein determination, the color of a dye changes from brown to blue when it binds to protein. Absorbance of light is measured. 

Final concentration can be estimated by comparison to ESA standards by SDS-PAGE analysis or Bradford protein determination assays... 

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... 

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. 

Protein determination Protein concentrations were determined according to Bradford [12] using BSA as a standard. 

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. 

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 obtain an active preparation it is essential to perform all steps without delay. Furthermore, avoid keeping synaptosomes resuspended for prolonged periods of time (e.g., use a fast dye-binding assay for protein determination that gives results in 10 min or less, such as that described by Bradford, 1976). The aliquoted synaptosomal pellets should not be resuspended until immediately before being used in the experiment. 

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

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. 

In this chapter, the preparation of synaptosomal plasma membranes using centrifugation techniques will be described in detail. The method here is based on that described previously by Kristjansson et al. (14) with only slight modifications. Section 3.1. outlines protocols for dissection and homogenization of the brain, indicating the parameters most important to obtain synaptosomal plasma membrane preparations of reproducibly high quality. Section 3.2. describes the subcellular fractionation procedure itself, and Section 3.3. outlines a protocol for assessment of yield of the synaptosomal plasma membranes employing a protein determination assay described by Bradford (15). 

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. 

OPH enzymes were purified in the presence of cobalt chloride as described (20). OPH fractions pooled after the first chromatography step (SP-Sepharose) were designated as impure OPH. The purity of OPH was determined by SDS-PAGE stained with Coomassie brilliant blue. The concentration of pure protein was estimated using the molar extinction coefficient for OPH ( 273 = 58,000 M cm ) and concentrations of impure samples were determined using the Bradford method of protein determination (28). 

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

Protein determination measurements were performed by Bradford s Method. Bradford reagent was prepared by mixing 25 ml phosphoric acid, 12.5 ml ethanol and 25 mg Coomassie Brilliant Blue (G-dye). The mixture was diluted to 50 ml with distilled water. During measurements, a solution of Bradford reagent was prepared by mixing 1 volume stock solution with 4 volumes of distilled water. 

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. 

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. 

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]. 

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). 

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. 

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. 

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