totalproteinlab - Chemistry 365 Biochemistry Laboratory...

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Chemistry 365 Biochemistry Laboratory Unit #3 Bradford Method for Total Protein Concentration: Selective Salting-Out of Proteins References : Bradford, M. M. Anal. Biochem. 1976 , 72 , p 248. Robyt, J.F.; White, B.J. Biochemical Techniques Theory and Practice ; Waveland: Prospect Heights, IL, 1990. Bollag, D.M.; Edelstein, S.J. Protein Methods ; Wiley-Liss: New York, 1991. The solubility of proteins is a function of the nature of the protein structure and form as well as the solution environment in which it exists. Factors such as pH, salt concentration, presence of organics, and presence of surfactants will affect the solubility. The procedure outlined here will illustrate the effect of ammonium sulfate concentration on the solubilities of different proteins in a mixture. Proteins extracted from mung bean sprouts will be precipitated in a 40% saturated ammonium sulfate solution. The precipitate is collected by centrifugation, redissolved, and the total protein concentration determined quantitatively by the Bradford protein assay. Solutions: 1) 0.05 M TRIS buffer, pH 7.2, 0.005 M NaCl. 2) Saturated solution of ammonium sulfate [528 g (NH 4 ) 2 SO 4 /L solution or 75 g + 100 mL water] with the pH adjusted to 7. 3) Standard protein solution (1.25 mg/mL bovine serum albumin in buffer #1) 4) Bradford Reagent Solution - 100 mg Coomassie Brilliant Blue G (65 -75% dye) in 50 mL of 95% ethanol.
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totalproteinlab - Chemistry 365 Biochemistry Laboratory...

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