TOTAL PROTEIN ASSAYS
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
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.
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.
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).
BRADFORD PROTEIN ASSAY PROCEDURE
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;
2.0 mL Assay reagent
Cover with Parafilm and gently invert several times
0.04 mL of protein standard solution, starting with the lowest protein
concentration and working up, or one of the samples to be assayed.
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;
0.2 mL Dye stock
Cover with Parafilm and gently invert several times to mix.
0.8 mL of one of the protein standard solutions or samples to be
assayed (containing <100 µg of protein for <50 µg/mL standards)
Follow the procedure described above for the standard assay
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1. Stoscheck, C. 1990 "Quantification of Protein" Methods in Enzymology
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.
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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