Quantitation of the amount of protein contained in a solution
can be conveniently determined using calorimetric methods. The
Bradford and the Lowry methods are the most frequently used and reliable procedures. The method
of choice is the Bradford method, which is easy and rapid to complete.
The Bradford method depends on quantitating the binding of a dye,
Coomassie Brilliant Blue, to an unknown protein and comparing
this binding to that of different amounts of a standard protein,
usually bovine serum albumin (BSA). It is designed to quantify
1 to 10 ug protein. Protein determinations in the range of 10
to 100 ug may be carried out by increasing the volume of the dye
solution 5-fold and using larger tubes.
3. Determine the A595 using a 1 cm pathlength microcuvette (1 ml) and make a standard
curve by plotting absorbancc at 595 nm versus protein concentration.
4. Determine the absorbance for the unknown and use the standard
curve to determine the concentration of protein in the unknown.
If the unknown protein concentration is too high, dilute the protein,
or generate another standard curve in a higher concentration range
(e.g., 10 to 100 ug).
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.
Brogdon, W.G. and Dickinson. C.M. 1983. A microassay system for
measuring esterase activity and protein concentration in small
samples and in high-pressure liquid chromatography eluate fractions.
Anal. Biochem. 131:499-503.
Cadman E., Bostwick, J.R., and Eichberg, J. 1979. Determination
of protein by a modified Lowry procedure in the presence of some
commonly used detergents. Anal. Biochem. 96:21-23.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.]. 1951.
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
Peterson, G.L. 1977. A simplification of the protein assay method
of Lowry et al. which is more generally applicable. Anal. Biochem.