Background & Theory

     Four spectroscopic methods are routinely used to determine the concentration of protein in a solution (1).  These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay (2), the Smith copper/bicinchoninic assay (3) and the Bradford dye assay (4).  Although one or more these methods is used routinely in almost every biochemical laboratory, none of the procedures are particularly convenient, for the reasons described below.

    The first, UV absorbance, requires that a pure protein with known extinction coefficient be used, in a solution free of interfering (UV absorbing) substances.  As an approximation, the protein concentration of a solution can be estimated by using either of the following equations;

A280 = 1 A (mL/cm mg) x [Conc.] (mg/mL) x 1 (cm)
 A205 = 31 A (mL/cm mg) x [Conc.] (mg/mL) x 1 (cm)

 Different proteins, however, have widely different extinction coefficients at both 280 and 205 nm, and concentration estimates obtained this way should be viewed with considerable skepticism.  Again, this assay requires that the protein solution be free of other UV absorbing substances, and that the measurements be made using a quartz cuvette.

     The Lowry and copper/bicinchoninic assays are based on reduction of Cu2+ to Cu1+ by amides.  Although this makes them potentially quite accurate, they require the preparation of several reagent solutions, which must be carefully measured and mixed during the assay.  This is followed by lengthy, precisely timed incubations at closely controlled, elevated temperatures, and then immediate absorbance measurements of the unstable solutions.  Both assays may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents (1). This requires that the assays also include a series of standard solutions, each with a different, known concentration of protein, but otherwise having the same composition as the sample solutions.

    The Bradford dye assay is based on the equilibrium between three forms of Coomassie Blue G dye.  Under strongly acid conditions, the dye is most stable as a doubly-protonated red form.  Upon binding to protein, however, it is most stable as an unprotonated, blue form.




    Red     <=>       Green      <=>           Blue         <=>           Blue-Protein
(470 nm)             (650 nm)                     (590 nm)                         (590 nm)
                  H+                                 H+
     The Bradford assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the assays described above.  Like the other assays, however, its response is prone to influence from non protein sources, particularly detergents, and becomes progressively more nonlinear at the high end of its useful protein concentration range.  The response is also protein dependent, and varies with the composition of the protein. (See examples)   These limitations make  protein standard solutions necessary.

    The reagents for all of the protein assays are widely available, and pre-weighed reagents (5), reagent mixtures (5), and reagent solutions (6,7) are available for the assays. Modifications of these assays, using proprietary solutions (8) or different protocols or formats are also described in the literature (9) or are commercially available (10).




 Dye stock - Coomassie Blue G (C.I.# 42655) (100 mg) is dissolved in 50 mL of methanol. (If turbid, the solution is treated with Norit (100 mg) and filtered through a glass-fiber filter.)  The solution is added to 100 mL of 85% H3PO4, and diluted to 200 mL with water.  The solution should be dark red, and have a pH of -0.01. The final reagent concentrations are 0.5 mg/mL Coomassie Blue G, 25% methanol, and 42.5% H 3PO4.  The solution is stable indefinitely in a dark bottle at 4°C.

 Assay reagent - The assay reagent is prepared by diluting 1 volume of the dye stock with 4 volumes of distilled H2O.  The solution should appear brown, and have a pH of 1.1.  It is stable for weeks in a dark bottle at 4°C.

 Protein Standards - Protein standards should be prepared in the same buffer as the samples to be assayed.  A convenient standard curve can be made using bovine serum albumin (BSA) with concentrations of 0, 250, 500, 1000, 1500, 2000 µg/mL for the standard assay, and 0, 10, 20, 30, 40, 50 µg/mL for the microassay.

Standard Protein Assay Procedure (200 - 2000 µg/mL protein):

Prepare six standard solutions (1 mL each) containing 0, 250, 500, 1000, 1500 and 2000 µg/mL BSA.
Set the spectrophotometer to collect the spectra over a wavelength range from 400 to 700 nm and over an absorbance range of 0 to 2 Absorbance units, and to overlay the collected spectra.
Use a 4 mL plastic cuvette filled with distilled water to blank the spectrophotometer over this wavelength range
    Empty the plastic cuvette into a test tube and shake out any remaining liquid.  Then add;     Cover with Parafilm and gently invert several times to mix
    Record the absorbance spectrum of the sample from 400 to 700 nm , and note the absorbance at 595 nm.
    Repeat the steps above for each of the protein standards and for the samples to be assayed.
    Examine the spectra of the standards and samples. If any spectrum has an absorbance at 595 nm greater than 2, or if any sample has an absorbance greater than the greatest absorbance for any of the standards, dilute the sample by a known amount and repeat the assay.  At one wavelength, approximately 575 nm, all of the spectra should have the same absorbance.  (Such an intersection is called an isosbestic point and is a defining characteristic of solutions containing the same total concentration of an absorbing species with two possible forms.)  If any spectrum does not intersect the other spectra at or near the isosbestic point, it should be adjusted or rejected and repeated.  (It is sometimes possible to adjust the baseline of a spectrum by determining the difference in absorbance at the isosbestic point from the absorbance of the other spectra at that wavelength, and adding the difference to the absorbance values at every wavelength of the spectrum. This correction works best at wavelengths close to the isosbestic point, and requires some discrimination by the spectroscopist.)
    Prepare a graph of Absorbance at 595 nm vs [Protein] for the protein standards.
    Examine the graphed points and decide if any should be rejected.  (Often a single point can be rejected without invalidating the standard curve, but if more than one point appears questionable the assay should be repeated.)  The Bradford assay gives a hyperbolic plot for absorbance versus protein concentration, but within a range of relatively low protein concentrations, the hyperbolic curve can be approximated reasonably well by a straight line. Use a best-fit straight line to fit the points if you feel it will give a good fit.  If not, draw a smooth curve that falls on or near each of the data points.
     To determine the protein concentration of a sample from it absorbance, use the standard curve to find the concentration of standard that would have the same absorbance as the sample.

Microassay Procedure (<50 µg/mL protein):

    Prepare five standard solutions (1 mL each) containing 0, 10, 20, 30,  40 and 50 µg/mL BSA
    To a 1.4 mL plastic cuvette, add;    Cover with Parafilm and gently invert several times to mix.
   Follow the procedure described above for the standard assay procedure.

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1. Stoscheck, C. 1990 "Quantification of Protein" Methods in Enzymology , 182:50-68.
2. Lowry, O. Rosebrough, A., Farr, A. and Randall, R. 1951 J. Biol. Chem . 193:265.
3. Smith, P. et al., (1985) Anal. Biochem. 150:76-85.
4. Bradford, 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.
5. anon. "Sigma Modified Lowry Protein Assay," Sigma Procedure No. P 5656.
6. anon. 1991 "BCA Protein Assay Reagent," Pierce catalog p. F-18.
7. anon. 1991 "Bio-Rad Protein Assay," Bio-Rad catalog p. 60.
8. anon. 1991 "Coomassie Plus Protein Assay Reagent" Pierce catalog p. F-22.
9. Cabib, E. and Polacheck, I. 1984 "Protein assay for dilute solutions." Methods in Enzymology, 104:318-328.
10. anon. 1991 "Fast Protein Assay," Pierce catalog  F-26.

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