Bradford protein assay binding assays

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. 

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

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

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. 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Another assay, the Bradford assay, also known as the Coomassie dye binding method, was first described in 1976 [19]. In an acidic environment, proteins will bind to Coomassie dye and cause a shift from the reddish brown color (465 nm) to the blue dye protein complex read at 595 nm. The development of the color is attributed to the presence of the basic amino acids arginine, lysine, and histidine. Van der Waals forces and hydrophobic interactions account for the dye binding and the number of Coomassie blue dye molecules bound is roughly proportional to the number of positive charges on the molecule. A protein molecular weight of about 3 kDA is required for successful color development. The Bradford assay dose response is nonlinear and this method demonstrates the greatest difference in reactivity with BSA compared to BGG. 

Analysis of cell components. Protein was determined with the Bradford dye binding assay using bovine serum albumin as standard ( )). Interference by Triton X100 was accounted for by ensuring that every sample had. 2% Triton. In order to determine the amount of unreleased protein from the sample pellets, all samples were treated for 5 minutes with IN NaOH at 100°C. 

Gringorten J L, Witt D P, Milne R E, et al. (1990). An in vitro system for testing Bacillus thuringiensis toxins The lawn assay. J. Invertebr. Pathol. 56 237-242. Bradford M (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 248-254. 

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. 

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

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... 

Washed cell pastes were diluted with fresh buffer using a ratio of 3 ml of buffer for each 1 g of cell paste. A French Cell Press operated at 16,000 lb/in was used to disrupt cells. The crude enzyme preparation was then centrifuged at 10,444 X g for 30 min at 0°C, and the supernatant was retained as a cell-free extract (CFE). This extract was carried through a series of activation and purification steps and the methlonlnase activity was assayed after each step using a modification of the method of Tanaka t al. (52). Protein was assayed by the dye-binding method of Bradford (62), and enzyme solutions were dialyzed in tubing prepared by procedures described by Brewers at al. (63). 

Various colorimetric methods are also available, based on non-specific dye binding to polypeptide chains, one of the more common being the Bradford assay. One drawback with such methods is that the actual colour intensity (absorbance) developed is not absolute, but depends on the specific protein. Calibration can therefore be a problem if accurate concentrations are required. 

The Bradford assay monitors a color change associated with the binding of Coomassie brilliant blue G-250 dye (CBBG) to protein in solution the unbound dye absorbs at 470 nm, while the complex absorbs at 595 nm. Unfortunately, not all proteins bind to CBBG equally, because of differing amino acid compositions. Therefore, not all proteins show the same response in a Bradford assay. 

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