The Bicinchoninic Acid (BCA) Assay for Protein Quantitation
John M. Walker
1. Introduction
The bicinchoninic acid (BCA) assay, first described by Smith et al. (1) is similar to
the Lowry assay, since it also depends on the conversion of Cu2+ to Cu+ under alkaline
conditions (see Chapter 2). The Cu+ is then detected by reaction with BCA. The two
assays are of similar sensitivity, but since BCA is stable under alkali conditions, this
assay has the advantage that it can be carried out as a one-step process compared to the
two steps needed in the Lowry assay. The reaction results in the development of an
intense purple color with an absorbance maximum at 562 nm. Since the production of
Cu+ in this assay is a function of protein concentration and incubation time, the protein
content of unknown samples may be determined spectrophotometrically by comparison
with known protein standards. A further advantage of the BCA assay is that it is
generally more tolerant to the presence of compounds that interfere with the Lowry
assay. In particular it is not affected by a range of detergents and denaturing agents
such as urea and guanidinium chloride, although it is more sensitive to the presence of
reducing sugars. Both a standard assay (0.1–1.0 mg protein/mL) and a microassay
(0.5–10 μg protein/mL) are described.
2. Materials
2.1. Standard Assay
1. Reagent A: sodium bicinchoninate (0.1 g), Na2CO3 · H2O (2.0 g), sodium tartrate (dihydrate)
(0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary, adjust
the pH to 11.25 with NaHCO3 or NaOH (see Note 1).
2. Reagent B: CuSO4 · 5H2O (0.4 g) in 10 mL of water (see Note 1).
3. Standard working reagent (SWR): Mix 100 vol of regent A with 2 vol of reagent B. The
solution is apple green in color and is stable at room temperature for 1 wk.
2.2. Microassay
1. Reagent A: Na2CO3 · H2O (0.8 g), NaOH (1.6 g), sodium tartrate (dihydrate) (1.6 g), made
up to 100 mL with water, and adjusted to pH 11.25 with 10 M NaOH.
2. Reagent B: BCA (4.0 g) in 100 mL of water.
3. Reagent C: CuSO4 · 5H2O (0.4 g) in 10 mL of water.
4. Standard working reagent (SWR): Mix 1 vol of reagent C with 25 vol of reagent B, then
add 26 vol of reagent A.
3. Methods
3.1. Standard Assay
1. To a 100-μL aqueous sample containing 10–100 μg protein, add 2 mL of SWR. Incubate
at 60°C for 30 min (see Note 2).
2. Cool the sample to room temperature, then measure the absorbance at 562 nm (see Note 3).
3. A calibration curve can be constructed using dilutions of a stock 1 mg/mL solution of
bovine serum albumin (BSA) (see Note 4).
3.2. Microassay
1. To 1.0 mL of aqueous protein solution containing 0.5–1.0 μg of protein/mL, add 1 mL of SWR.
2. Incubate at 60°C for 1 h.
3. Cool, and read the absorbance at 562 nm.
4. Notes
1. Reagents A and B are stable indefinitely at room temperature. They may be purchased
ready prepared from Pierce, Rockford, IL.
2. The sensitivity of the assay can be increased by incubating the samples longer. Alternatively,
if the color is becoming too dark, heating can be stopped earlier. Take care to treat
standard samples similarly.
3. Following the heating step, the color developed is stable for at least 1 h.
4. Note, that like the Lowry assay, response to the BCA assay is dependent on the amino acid
composition of the protein, and therefore an absolute concentration of protein cannot be
determined. The BSA standard curve can only therefore be used to compare the relative
protein concentration of similar protein solutions.
5. Some reagents interfere with the BCA assay, but nothing like as many as with the Lowry
assay (see Table 1). The presence of lipids gives excessively high absorbances with this
assay (2). Variations produced by buffers with sulfhydryl agents and detergents have been
described (3).
6. Since the method relies on the use of Cu2+, the presence of chelating agents such as EDTA
will of course severely interfere with the method. However, it may be possible to overcome
such problems by diluting the sample as long as the protein concentration remains
sufficiently high to be measurable. Similarly, dilution may be a way of coping with any
agent that interferes with the assay (see Table 1). In each case it is of course necesary to
run an appropriate control sample to allow for any residual color development. A modification
of the assay has been described that overcomes lipid interference when measuring
lipoprotein protein content (4).
7. A modification of the BCA assay, utilizing a microwave oven, has been described that
allows protein determination in a matter of seconds (see Chapter 5).
8. A method has been described for eliminating interfering compounds such as thiols and
reducing sugars in this assay. Proteins are bound to nylon membranes and exhaustively
washed to remove interfering compounds; then the BCA assay is carried out on the membrane-
bound protein (5).
9. A comparison of the BCA, Lowry and Bradford assays for analyzing gylcosylated and
non-glycosylated proteins have been made (6). Significant differences wee observed
between the assays for non-glycosylated proteins with the BCA assay giving results closest
to those from amino acid analysis. Glycosylated proteins were underestimated by the
Bradford the method and overestimated by the BCA and Lowry methods. The results
suggest a potential interference of protein glycosylation with colorimetric assays.
10. A modification of this assay for analysis complex samples, which involves removing contaminants
from the protein precipitate with 1 M HCl has been reported (7).
References
1. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano,
M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement
of protein using bicinchoninic acid. Analyt. Biochem. 150, 76–85.
2. Kessler, R. J. and Fanestil, D. D. (1986) Interference by lipids in the determination of
protein using bicinchoninic acid. Analyt. Biochem. 159, 138–142.
3. Hill, H. D. and Straka, J. G. (1988) Protein determination using bicinchoninic acid in the
presence of sulfhydryl reagents. Analyt. Biochem. 170, 203–208.
4. Morton, R. E. and Evans, T. A. (1992) Modification of the BCA protein assay to eliminate
lipid interference in determining lipoprotein protein content. Analyt. Biochem. 204,
332–334.
5. Gates, R. E. (1991) Elimination of interfering substances in the presence of detergent in
the bicinchoninic acid protein assay. Analyt. Biochem. 196, 290–295.
6. Fountoulakis, M., Juranville, J. F., and Manneberg, M. (1992) Comparison of the coomassie
brilliant blue, bicinchoninic acid and lowry quantitation assays, using nonglycosylated and
glycosylated proteins. J. Biochem. Biophys. Meth. 24, 265–274.
7. Schoel, B., Welzel, M., and Kaufmann, S. H. E. (1995) Quantification of protein in dilute
and complex samples–modification of the bicinchoninic acid assay. J. Biochem. Biophys.
Meth. 30, 199–206.
