protein estimation
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• Determination of quantity of protein in given solution
• Colorimetric protein assay• Absorbance path length * Concentration• Absorbance Concentration (Path length:
Constant)
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Biuret reaction + Reduction of Phosphomolybdate and Phosphotungstate from Folin Ciocateu’s (Phenol) reagent
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• Reagent B : 1 : 5 Phenol reagent (phosphomolybdic-phosphotungstic acid)
10 ml of Folin Ciocalteau reagent dilute with 50 ml with DW
Highly SensitiveReagents are stableMore concentratedCan be used for both Normal & Micro assay
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• First protein is reacted with alkaline cupric sulfate in the presence of tartarate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper.
• Second a i.e. Folin-phenol reagent is added, it reduced to give intense blue color.
• Reaction involves reduction of the Folin reagent and oxidation of aromatic residues.
• (Conversion of Cupric Cu+2 to Cuprous Cu+ ions, Read the blue colour developed, at 610 nm for colorimeter)
• Specificity: The Lowry method is best used with protein concentrations of 0.01-1.0 mg/mL
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• Bradford, M.M. (1976). Anal. Biochem. 72 , 248-254.
• Smith, P.K., et al. (1985). Anal Biochem. 150 , 76-85.
• Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc.
• Krohn, R.I. (2001). The Colorimetric Determination of Total Protein, Current Protocols in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc.
• Lowry, O.H., et al. (1951). J Biol Chem. 193 , 265-275.
• Legler, G., et al. (1985). Anal. Biochem. 150 , 278-287.
• Coligan, J.E., et al. , Eds. (2002). Electrophoresis, In Current Protocols in Protein Science, pp. 10.0.1-10.4.36. John Wiley and Sons, Inc. New York.
• Bollag, D.M., Rozycki, M.D. and Edelstein, S.J. (2002). Protein Methods, 2nd ed. Wiley-Liss, Inc. New York.
• Hames, B.D. and Rickwood, D. Eds. (1990) Gel Electrophoresis of Proteins: a Practical Approach, 2nd ed. Oxford University Press, New York.