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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 Cu 2 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 lag protein/mL) are described.

2. Materials

2.1. Standard Assay

1. Reagent A: sodium bicinchoninate (0.1 g), Na2CO 3 H20 (2.0 g), sodium tartrate (dihy9 drate) (0.16 g), NaOH (0.4 g), NaHCO 3 (0.95 g), made up to 100 mL. If necessary, adjust the pH to 11.25 with NaHCO 3 or NaOH (see Note 1). 2. Reagent B: CuSO 4 5H20 (0.4 g) in 10 mL of water (see Note 1). 9 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: Na2CO 3 H20 (0.8 g), NaOH (1.6 g), sodium tartrate (dihydrate) (1.6 g), made 9 up to 100 mL with water, and adjusted to pH 11.25 with 10MNaOH. 2. Reagent B: BCA (4.0 g) in 100 mL of water. 3. Reagent C: CuSO4" 5H20 (0.4 g) in 10 mL of water.
From: The Protein Protocols Handbook Edited by: J. M. Walker Humana Press Inc., Totowa, NJ

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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 100-1aL aqueous sample containing 10-100 ~g protein, add 2 mL of SWR. Incubate at 60~ 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 ~tg ofprotein/mL, add 1 mL of SWR. 2. Incubate at 60~ 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 Cu 2+, 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).

BCA for Protein Quantitation

Table 1 Effect of Selected Potential Interfering Compounds a
BCA assay (~tg BSA found) Sample (50 lag BSA) in the following 50 jag BSA in water (reference) 0.1NHC1 0.1NNaOH 0.2% Sodium azide 0.02% Sodium azide 1.0M Sodium chloride 100 mM EDTA (4 Na) 50 mM EDTA (4 Na) 10 mM EDTA (4 Na) 50 mM EDTA (4 Na), pH 11.25 4.0M Guanidine HC1 3.0MUrea 1.0%Triton X- 100 1.0% SDS (lauryl) 1.0% Brij 35 1.0% Lubrol 1.0% Chaps 1.0% Chapso 1.0% Octyl glucoside 40.0% Sucrose 10.0% Sucrose 1.0% Sucrose 100 mM Glucose 50 mM Glucose 10 mM Glucose 0.2M Sorbitol 0.2M Sorbitol, pH 11.25 1.0M Glycine 1.0M Glycine, pH 11 0.5MTris 0.25M Yris 0.1MTris 0.25M Tris, pH 11.25 20.0% Ammonium sulfate 10.0% Ammonium sulfate 3.0% Ammonium sulfate 10.0% Ammonium sulfate, pH 11 2.0M Sodium acetate, pH 5.5 0.2M Sodium acetate, pH 5.5 1.0M Sodium phosphate 0.1M Sodium phosphate 0.1M Cesium bicarbonate Water blank corrected Interference blank corrected Lowry assay (lag BSA found) Water blank corrected Interference blank corrected

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50.00 50.70 50.80 49.00 49.40 51.10 50.90 51.10 51.00 51.30 51.10 No color 28.00 29.40 48.80 49.10 31.50 32.80 48.30 46.90 51.30 50.10 50.20 49.80 49.20 48.90 51.00 50.90 50.70 50.70 49.90 49.50 51.80 51.00 50.90 50.80 55.40 48.70 52.50 50.50 51.30 51.20 245.00 57.10 144.00 47.70 70.00 49.10 42.90 37.80 40.70 36.20 No color 50.70 48.90 36.20 32.90 46.60 44.00 50.80 49.60 52.00 50.30 5.60 1.20 16.00 12.00 44.90 42.00 48.10 45.20 35.50 34.50 50.80 50.40 37.10 36.20 50.80 50.40 49.50 49.70

50.00 44.20 43.80 50.60 50.60 49.20 49.00 49.50 49.60 50.20 50.10 38.50 5.10 96.70 6.80 33.60 12.70 72.30 5.00 Precipitated 53.20 45.00 Precipitated Precipitated Precipitated Precipitated Precipitated Precipitated Precipitated 4.90 28.90 42.90 41.10 48.40 48.10 68.10 61.70 62.70 58.40 52.60 51.20 63.70 31.00 68.60 26.60 7.30 7.70 32.50 27.90 10.20 8.80 27.90 28.10 38.90 38.90 40.80 40.80 Precipitated Precipitated 21.40 21.20 32.80 32.60 5.40 3.30 47.60 47.50 5.30 7.30 46.60 46.60 Precipitated

aReproduced from ref. 1 with permission from Academic Press Inc.

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References

Walker

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. AnaL Biochem. 150, 76-85. 2. Kessler, R. J. and Fanestil, D. D. (1986) Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem. 159, 138-142. 3. Hill, H. D. and Straka, J. G. (1988) Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents. Anal. 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. Anal. Biochem. 204, 332-334. 5. Gates, R. E. (1991) Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay. Anal. Biochem. 196, 290-295.