Monday, March 21, 2011

PROTEIN DETERMINATION

1. UV Absorbance for Protein determination

1.1. Near UV Absorbance (280 nm)

Quantitation of the amount of protein in a solution is possible in a simple spectrom­eter. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). There­fore the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5–1.5 [1]). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be deter­mined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.

1.2. Far UV Absorbance

The peptide bond absorbs strongly in the far UV with a maximum at about 190 nm. This very strong absorption of proteins at these wavelengths has been used in protein determination. Because of the difficulties caused by absorption by oxygen and the low output of conventional spectrophotometers at this wavelength, measurements are more conveniently made at 205 nm, where the absorbance is about half that at 190 nm. Most proteins have extinction coefficients at 205 nm for a 1 mg/mL solution of 30–35 and between 20 and 24 at 210 nm (2).

Various side chains, including those of Trp, Phe, Tyr, His, Cys, Met, and Arg (in that descending order), make contributions to the A205 (3).

The advantages of this method include simplicity and sensitivity. As in the method outlined in Subheading 3.1. the sample is recoverable and in addition there is little variation in response between different proteins, permitting near-absolute determina­tion of protein. Disadvantages of this method include the necessity for accurate calibra­tion of the spectrophotometer in the far UV. Many buffers and other components, such as heme or pyridoxal groups, absorb strongly in this region.

2. Materials

1. 0.1 M K2SO4 (pH 7.0).

2. 5 mM potassium phosphate buffer, pH 7.0.

3. Nonionic detergent (0.01% Brij 35)

4. Guanidinium-HCl.

5. 0.2-µm Millipore (Watford, UK) filter.

6. UV-visible spectrometer: The hydrogen lamp should be selected for maximum intensity at the particular wavelength.

7. Cuvets, quartz, for less tha 215 nm.


3. Methods

3.1. Estimation of Protein by Near UV Absorbance (280 nm)

1. A reliable spectrophotometer is necessary. The protein solution must be diluted in the buffer to a concentration that is well within the accurate range of the instrument (see Notes 1 and 2).

2. The protein solution to be measured can be in a wide range of buffers, so it is usually no problem to find one that is appropriate for the protein which may already be in a particular buffer required for a purification step or assay for enzyme activity, for example (see Notes 3 and 4).

3. Measure the absorbance of the protein solution at 280 nm, using quartz cuvets or cuvets that are known to be transparent to this wavelength, filled with a volume of solution suffi­cient to cover the aperture through which the light beam passes.

4. The value obtained will depend on the path length of the cuvet. If not 1 cm, it must be adjusted by the appropriate factor. The Beer-Lambert law states that:

a. A (absorbance) = ε c l (1)

6. where ε= extinction coefficient, c = concentration in mol/L and l = optical path length in cm. Therefore, if ε is known, measurement of A gives the concentration directly; ε is normally quoted for a 1-cm path length.

7. The actual value of UV absorbance for a given protein must be determined by some abso­lute method, e.g., calculated from the amino acid composition, which can be determined by amino acid analysis (4). The UV absorbance for a protein is then calculated according to the following formula:

i. A280 (1 mg/mL) = (5690nw + 1280ny + 120nc)/M (2)

8. where nw, ny, and nc are the numbers of Trp, Tyr, and Cys residues in the polypeptide of mass M and 5690, 1280 and 120 are the respective extinction coefficients for these resi­dues (see Note 5).

3.2. Estimation of Protein by Far UV Absorbance

1. The protein solution is diluted with a sodium chloride solution (0.9% w/v) until the absor­bance at 215 nm is less than 1.5 (see Notes 1 and 6).

2. Alternatively, dilute the sample in another non-UV-absorbing buffer such as 0.1 M K2SO4, containing 5 mM potassium phosphate buffer adjusted to pH 7.0 (see Note 6).

3. Measure the absorbances at the appropriate wavelengths (either A280 and A205, or A225 and A215, depending on the formula to be applied), using a spectrometer fitted with a hydrogen lamp that is accurate at these wavelengths, using quartz cuvets filled with a volume of solution sufficient to cover the aperture through which the light beam passes (details in Subheading 3.1.).

4. The A205 for a 1 mg/mL solution of protein (A2051 mg/mL) can be calculated within ±2%, according to the empirical formula proposed by Scopes (2) (see Notes 7–10):

A2051 mg/mL = 27 + 120 (A280/A205) (3)

5. Alternatively, measurements may be made at longer wavelengths (5):

Protein concentration (µg/mL) = 144 (A215 – A225) (4)

The extinction at 225 nm is subtracted from that at 215 nm; the difference multiplied by 144 gives the protein concentration in the sample in µg/mL. With a particular protein under specific conditions accurate measurements of concentration to within 5 µg/L are possible.

4. Notes

1. It is best to measure absorbances in the range 0.05–1.0 (between 10 and 90% of the inci­dent radiation). At around 0.3 absorbance (50% absorption), the accuracy is greatest.

2. Bovine serum albumin is frequently used as a protein standard; 1 mg/mL has an A280 of 0.66.

3. If the solution is turbid, the apparent A280 will be increased by light scattering. Filtration (through a 0.2-µm Millipore filter) or clarification of the solution by centrifugation can be carried out. For turbid solutions, a convenient approximate correction can be applied by subtracting the A310 (proteins do not normally absorb at this wavelength unless they con­tain particular chromophores) from the A280.

4. At low concentrations, protein can be lost from solution by adsorption on the cuvet; the high ionic strength helps to prevent this. Inclusion of a nonionic detergent (0.01% Brij 35) in the buffer may also help to prevent these losses.

5. The presence of nonprotein chromophores (e.g., heme, pyridoxal) can increase A280. If nucleic acids are present (which absorb strongly at 260 nm), the following formula can be applied. This gives an accurate estimate of the protein content by removing the contribu­tion to absorbance by nucleotides at 280 nm, by measuring the A260 which is largely owing to the latter (6).

Protein (mg/mL) = 1.55 A280 – 0.76 A260 (5)

Other formulae (using similar principles of absorbance differences) employed to deter­mine protein in the possible presence of nucleic acids are the following (7,8):

Protein (mg/mL) = (A235 – A280)/2.51 (6)

Protein (mg/mL) = 0.183 A230 – 0.075.8 A260 (7)

6. Protein solutions obey Beer-Lambert’s Law at 215 nm provided the absorbance is less than 2.0.

7. Strictly speaking, this value applies to the protein in 6 M guanidinium-HCl, but the value in buffer is generally within 10% of this value, and the relative absorbances in guani-dinium-HCl and buffer can be easily determined by parallel dilutions from a stock solution.

8. Sodium chloride, ammonium sulfate, borate, phosphate, and Tris do not interfere, whereas

0.1 M acetate, succinate, citrate, phthalate, and barbiturate show high absorption at 215 nm.

9. The absorption of proteins in the range 215–225 nm is practically independent of pH between pH values 4–8.

10. The specific extinction coefficient of a number of proteins and peptides at 205 nm and 210 nm (3) has been determined. The average extinction coefficient for a 1 mg/mL solu­tion of 40 serum proteins at 210 nm is 20.5 ± 0.14. At this wavelength, a protein concen­tration of 2 µg/mL gives A = 0.04 (5).

References

1. Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Analyt. Biochem. 68, 465–484.

2. Scopes, R. K. (1974) Measurement of protein by spectrometry at 205 nm. Analyt. Biochem. 59, 277–282.

3. Goldfarb, A. R., Saidel, L. J., and Mosovich, E. (1951) The ultraviolet absorption spectra of proteins. J. Biol. Chem. 193, 397–404.

4. Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Analyt. Biochem. 182, 319–326.

5. Waddell, W. J. (1956) A simple UV spectrophotometric method for the determination of protein. J. Lab. Clin. Med. 48, 311–314.

6. Layne, E. (1957) Spectrophotornetric and turbidimetric methods for measuring proteins. Meth. Enzymol. 3, 447–454.

7. Whitaker, J. R. and Granum, P. E. (1980) An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Analyt. Biochem. 109, 156–159.

8. Kalb, V. F. and Bernlohr, R. W. (1977). A new spectrophotometric assay for protein in cell extracts. Analyt. Biochem. 82, 362–371.

2. The Lowry Method for Protein Quantitation

Jakob H. Waterborg

1. Introduction

The most accurate method of determining protein concentration is probably acid hydrolysis followed by amino acid analysis. Most other methods are sensitive to the amino acid composition of the protein, and absolute concentrations cannot be obtained (1). The procedure of Lowry et al. (2) is no exception, but its sensitivity is moderately constant from protein to protein, and it has been so widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous absolute determina­tion in almost all circumstances in which protein mixtures or crude extracts are involved.

The method is based on both the Biuret reaction, in which the peptide bonds of proteins react with copper under alkaline conditions to produce Cu+, which reacts with the Folin reagent, and the Folin–Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/mL, and is best used on solutions with concentrations in the range 0.01–1.0 mg/mL of protein.

2. Materials

1. Complex-forming reagent: Prepare immediately before use by mixing the following stock solutions in the proportion 100:1:1 (by vol), respectively: Solution A: 2% (w/v) Na2CO3 in distilled water. Solution B: 1% (w/v) CuSO4·5H2O in distilled water. Solution C: 2% (w/v) sodium potassium tartrate in distilled water.

2. 2 N NaOH.

3. Folin reagent (commercially available): Use at 1 N concentration.

4. Standards: Use a stock solution of standard protein (e.g., bovine serum albumin fraction V) containing 2 mg/mL protein in distilled water, stored frozen at –20°C. Prepare standards by diluting the stock solution with distilled water as follows: Stock solution (µL) 0 2.5 512.5 25 50125 250 500 Water (µL) 500 498 495 488 475 450 375 250 0 Protein conc. (µg/mL) 0 10 20 50 100 200 500 1000 2000

3. Method

1. To 0.1 mL of sample or standard (see Notes 1–4), add 0.1 mL of 2 N NaOH. Hydrolyze at 100°C for 10 min in a heating block or boiling water bath.

2. Cool the hydrolysate to room temperature and add 1 mL of freshly mixed complex-form-ing reagent. Let the solution stand at room temperature for 10 min (see Notes 5 and 6).

3. Add 0.1 mL of Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 30–60 min (do not exceed 60 min) (see Note 7).

4. Read the absorbance at 750 nm if the protein concentration was below 500 µg/mL or at 550 nm if the protein concentration was between 100 and 2000 µg/mL.

5. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations (see Notes 8–13).

4. Notes

1. If the sample is available as a precipitate, then dissolve the precipitate in 2 N NaOH and hydrolyze as described in Subheading 3, step 1. Carry 0.2-mL aliquots of the hydrolyzate forward to Subheading 3, step 2.

2. Whole cells or other complex samples may need pretreatment, as described for the Burton assay for DNA (3). For example, the perchloroacetic acid (PCA)/ethanol precipitate from extraction I may be used directly for the Lowry assay, or the pellets remaining after the PCA hydrolysis step (Subheading 3, step 3 of the Burton assay) may be used for Lowry. In this latter case, both DNA and protein concentration may be obtained from the same sample.

3. Peterson (4) has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins in dilute solution. Peterson’s precipitation step is as follows:

a. Add 0.1 mL of 0.15% deoxycholate to 1.0 mL of protein sample.

b. Vortex-mix, and stand at room temperature for 10 min.

c. Add 0.1 mL of 72% trichloroacetic acid (TCA), vortex-mix, and centrifuge at 1000– 3000g for 30 min.

d. Decant the supernatant and treat the pellet as described in Note 1.

4. Detergents such as sodium dodecyl sulfate (SDS) are often present in protein prepara­tions, added to solubilize membranes or remove interfering substances (5–7). Protein pre­cipitation by TCA may require phosphotungstic acid (PTA) (6) for complete protein recovery:

a. Add 0.2 mL of 30% (w/v) TCA and 6% (w/v) PTA to 1.0 mL of protein sample.

b. Vortex-mix, and stand at room temperature for 20 min.

c. Centrifuge at 2000g and 4°C for 30 min.

d. Decant the supernatant completely and treat the pellet as described in Note 1.

5. The reaction is very pH dependent, and it is therefore important to maintain the pH between 10 and 10.5. Therefore, take care when analyzing samples that are in strong buffer outside this range.

6. The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.

7. The vortex-mixing step is critical for obtaining reproducible results. The Folin reagent is reactive only for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.

8. The assay is not linear at higher concentrations. Ensure that you are analyzing your sample on the linear portion of the calibration curve.

9. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.

10. One disadvantage of the Lowry method is the fact that a range of substances interferes with this assay, including buffers, drugs, nucleic acids, and sugars. (The effect of some of these agents is shown in Table 1 in Chapter 3.) In many cases, the effects of these agents can be minimized by diluting them out, assuming that the protein concentration is suffi­ciently high to still be detected after dilution. When interfering compounds are involved, it is, of course, important to run an appropriate blank. Interference caused by detergents, sucrose, and EDTA can be eliminated by the addition of SDS (5) and a precipitation step (see Note 4).

11. Modifications to this basic assay have been reported that increase the sensitivity of the reaction. If the Folin reagent is added in two portions, vortex-mixing between each addi­tion, a 20% increase in sensitivity is achieved (8). The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50% (9).

12. The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein(s) (see Introduction). There­fore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay. It must be appreciated; therefore, that using bovine serum albumin (BSA) (or any other protein for that matter) as a standard gives only an approximate measure of the protein concentration. The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve. The most accurate way to determine the concentration of any protein solution is amino acid analysis.

13. A means of speeding up this assay using raised temperatures (10) or a microwave oven (see Chapter 5) has been described.

References

1. Sapan, C. V., Lundblad, R. L., and Price, N. C. (1999) Colorimetric protein assay tech­niques. Biotechnol. Appl. Biochem. 29, 99–108.

2. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measure­ment with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

3. Waterborg, J. H. and Matthews, H. R. (1984) The Burton assay for DNA, in Methods in Molecular Biology, Vol. 2: Nucleic Acids (Walker, J. M., ed.), Humana Press, Totowa, NJ, pp. 1–3.

4. Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95–121.

5. Markwell, M.A.K., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Protein determi­nation in membrane and lipoprotein samples. Methods Enzymol. 72, 296–303.

6. Yeang, H. Y., Yusof, F., and Abdullah, L. (1998) Protein purification for the Lowry assay: acid precipitation of proteins in the presence of sodium dodecyl sulfate and other biologi­cal detergents. Analyt. Biochem. 265, 381–384.

7. Chang, Y. C. (1992) Efficient precipitation and accurate quantitation of detergent-solubi-lized membrane proteins. Analyt. Biochem. 205, 22–26.

8. Hess, H. H., Lees, M. B., and Derr, J. E. (1978) A linear Lowry-Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins. Analyt. Biochem. 85, 295–300.

9. Larson, E., Howlett, B., and Jagendorf, A. (1986) Artificial reductant enhancement of the Lowry method for protein determination. Analyt. Biochem. 155, 243–248.

10. Shakir, F. K., Audilet, D., Drake, A. J., and Shakir, K. M. (1994) A rapid protein determi­nation by modification of the Lowry procedure. Analyt. Biochem. 216, 232–233.

3. 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 mem-brane-bound protein (5).

Table 1

Effect of Selected Potential Interfering Compoundsa



BCA assay Lowry assay (µg BSA found) (µg BSA found)

Water

Interference

Water Interference

Sample (50 µg BSA)

blank

blank

blank blank

in the following

corrected

corrected

corrected corrected

50 µg BSA in water (reference)

50.00

50.00 —

0.1 N HCl

50.70

50.80

44.20 43.80

0.1 N NaOH

49.00

49.40

50.60 50.60

0.2% Sodium azide

51.10

50.90

49.20 49.00

0.02% Sodium azide

51.10

51.00

49.50 49.60

1.0 M Sodium chloride

51.30

51.10

50.20 50.10

100 mM EDTA (4 Na)

No color

138.50 5.10

50 mM EDTA (4 Na)

28.00

29.40

96.70 6.80

10 mM EDTA (4 Na)

48.80

49.10

33.60 12.70

50 mM EDTA (4 Na), pH 11.25

31.50

32.80

72.30 5.00

4.0 M Guanidine HCl

48.30

46.90

Precipitated

3.0 M Urea

51.30

50.10

53.20 45.00

1.0%Triton X-100

50.20

49.80

Precipitated

1.0% SDS (lauryl)

49.20

48.90

Precipitated

1.0% Brij 35

51.00

50.90

Precipitated

1.0% Lubrol

50.70

50.70

Precipitated

1.0% Chaps

49.90

49.50

Precipitated

1.0% Chapso

51.80

51.00

Precipitated

1.0% Octyl glucoside

50.90

50.80

Precipitated

40.0% Sucrose

55.40

48.70

4.90 28.90

10.0% Sucrose

52.50

50.50

42.90 41.10

1.0% Sucrose

51.30

51.20

48.40 48.10

100 mM Glucose

245.00

57.10

68.10 61.70

50 mM Glucose

144.00

47.70

62.70 58.40

10 mM Glucose

70.00

49.10

52.60 51.20

0.2 M Sorbitol

42.90

37.80

63.70 31.00

0.2 M Sorbitol, pH 11.25

40.70

36.20

68.60 26.60

1.0 M Glycine

No color

7.30 7.70

1.0 M Glycine, pH 11

50.70

48.90

32.50 27.90

0.5 M Tris

36.20

32.90

10.20 8.80

0.25 M Tris

46.60

44.00

27.90 28.10

0.1 M Tris

50.80

49.60

38.90 38.90

0.25 M Tris, pH 11.25

52.00

50.30

40.80 40.80

20.0% Ammonium sulfate

5.60

1.20

Precipitated

10.0% Ammonium sulfate

16.00

12.00

Precipitated

3.0% Ammonium sulfate

44.90

42.00

21.20 21.40

10.0% Ammonium sulfate, pH 11

48.10

45.20

32.60 32.80

2.0 M Sodium acetate, pH 5.5

35.50

34.50

5.40 3.30

0.2 M Sodium acetate, pH 5.5

50.80

50.40

47.50 47.60

1.0 M Sodium phosphate

37.10

36.20

7.30 5.30

0.1 M Sodium phosphate

50.80

50.40

46.60 46.60

0.1 M Cesium bicarbonate

49.50

49.70

Precipitated

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

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) Measurementof 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.

4. The Bradford Method for Protein Quantitation

Nicholas J. Kruger

1. Introduction

A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. An assay originally described by Bradford (1) has become the preferred method for quantifying protein in many laboratories. This tech­nique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common rea­gents and nonprotein components of biological samples (see Note 1).

The Bradford assay relies on the binding of the dye Coomassie Blue G250 to pro­tein. Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82, and 12.4 (2). Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maxi­mum at 590 nm. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absor­bance of the solution at 595 nm (see Note 2).

The dye appears to bind most readily to arginyl and lysyl residues of proteins (3,4). This specificity can lead to variation in the response of the assay to different proteins, which is the main drawback of the method (see Note 3). The original Bradford assay shows large variation in response between different proteins (5–7). Several modifica­tions to the method have been developed to overcome this problem (see Note 4). How­ever, these changes generally result in a less robust assay that is often more susceptible to interference by other chemicals. Consequently, the original method devised by Bradford remains the most convenient and widely used formulation. Two types of assay are described here: the standard assay, which is suitable for measuring between 10 and 100 µg of protein, and the microassay, which detects between 1 and 10 µg of protein. The latter, although more sensitive, is also more prone to interference from other com­pounds because of the greater amount of sample relative to dye reagent in this form of the assay.

2. Materials

1. Reagent: The assay reagent is made by dissolving 100 mg of Coomassie Blue G250 in 50 mL of 95% ethanol. The solution is then mixed with 100 mL of 85% phosphoric acid and made up to 1 L with distilled water (see Note 5).

The reagent should be filtered through Whatman no. 1 filter paper and then stored in an amber bottle at room temperature. It is stable for several weeks. However, during this time dye may precipitate from solution and so the stored reagent should be filtered before use.

2. Protein standard (see Note 6). Bovine γ-globulin at a concentration of 1 mg/mL (100 µg/mL for the microassay) in distilled water is used as a stock solution. This should be stored frozen at –20oC. Since the moisture content of solid protein may vary during storage, the precise concentration of protein in the standard solution should be determined from its absorbance at 280 nm. The absorbance of a 1 mg/mL solution of γ-globulin, in a 1-cm light path, is 1.35. The corresponding values for two alternative protein standards, bovine serum albumin and ovalbumin, are 0.66 and 0.75, respectively.

3. Plastic and glassware used in the assay should be absolutely clean and detergent free. Quartz (silica) spectrophotometer cuvettes should not be used, as the dye binds to this material. Traces of dye bound to glassware or plastic can be removed by rinsing with methanol or detergent solution.

3. Methods

3.1. Standard Assay Method

1. Pipet between 10 and 100 µg of protein in 100 µL total volume into a test tube. If the approximate sample concentration is unknown, assay a range of dilutions (1, 1:10, 1:100, 1:1000). Prepare duplicates of each sample.

2. For the calibration curve, pipet duplicate volumes of 10, 20, 40, 60, 80, and 100 µL of 1 mg/mL γ-globulin standard solution into test tubes, and make each up to 100 µL with distilled water. Pipet 100 µL of distilled water into a further tube to provide the reagent blank.

3. Add 5 mL of protein reagent to each tube and mix well by inversion or gentle vortex-mixing. Avoid foaming, which will lead to poor reproducibility.

4. Measure the A595 of the samples and standards against the reagent blank between 2 min and 1 h after mixing (see Note 7). The 100 µg standard should give an A595 value of about 0.4. The standard curve is not linear, and the precise absorbance varies depending on theage of the assay reagent. Consequently, it is essential to construct a calibration curve for each set of assays (see Note 8).

3.2. Microassay Method

This form of the assay is more sensitive to protein. Consequently, it is useful when the amount of the unknown protein is limited (see also Note 9).

1. Pipet duplicate samples containing between 1 and 10 µg in a total volume of 100 µL into 1.5-mL polyethylene microfuge tubes. If the approximate sample concentration is unknown, assay a range of dilutions (1, 1:10, 1:100, 1:1000).

2. For the calibration curve, pipet duplicate volumes of 10, 20, 40, 60, 80, and 100 µL of 100 µg/mL γ-globulin standard solution into microfuge tubes, and adjust the volume to 100 µL with water. Pipet 100 µL of distilled water into a tube for the reagent blank.

3. Add 1 mL of protein reagent to each tube and mix gently, but thoroughly.

Fig. 1. Variation in the response of proteins in the Bradford assay.

The extent of protein–dye complex formation was determined for bovine serum albumin (), γ-globulin (), and ovalbu­min () using the microassay. Each value is the mean of four determinations. For each set of measurements the standard error was less than 5% of the mean value. The data allow comparisons to be made between estimates of protein content obtained using these protein standards.

4. Measure the absorbance of each sample between 2 and 60 min after addition of the protein reagent. The A595 value of a sample containing 10 µg γ-globulin is 0.45. Figure 1 shows the response of three common protein standards using the microassay method.

4. Notes

1. The Bradford assay is relatively free from interference by most commonly used biochemi­cal reagents. However, a few chemicals may significantly alter the absorbance of the rea­gent blank or modify the response of proteins to the dye (Table 1). The materials that are most likely to cause problems in biological extracts are detergents and ampholytes (3,8). These can be removed from the sample solution by gel filtration, dialysis, or precipitation of protein with calcium phosphate (9,10). Alternatively, they can be included in the rea­gent blank and calibration standards at the same concentration as that found in the sample. The presence of base in the assay increases absorbance by shifting the equilibrium of the free dye toward the anionic form. This may present problems when measuring protein content in concentrated basic buffers (3). Guanidine hydrochloride and sodium ascorbate compete with dye for protein, leading to underestimation of the protein content (3).

2. Binding of protein to Coomassie Blue G250 may shift the absorbance maximum of the blue ionic form of the dye from 590 nm to 620 nm (2). It might, therefore, appear more sensible to measure the absorbance at the higher wavelength. However, at the usual pH of the assay, an appreciable proportion of the dye is in the green form (λmax = 650 nm) which interferes with absorbance measurement of the dye–protein complex at 620 nm. Measure­ment at 595 nm represents the best compromise between maximizing the absorbence due to the dye–protein complex while minimizing that due to the green form of the free dye (2–4; but see also Note 9).

Table 1


Effects of Common Reagents on the
Bradford Assay



Absorbance at 600 nm

Compound

Blank

5 mg Immunoglobulin

Control

0.005

0.264

0.02% SDS

0.003

0.250

0.1% SDS

0.042a

0.059a

0.1% Triton

0.000

0.278

0.5% Triton

0.051a

0.311a

1 M 2-Mercaptoethanol

0.006

0.273

1 M Sucrose

0.008

0.261

4 M Urea

0.008

0.261

4 M NaCl

–0.015

0.207a

Glycerol

0.014

0.238a

0.1 M HEPES, pH 7.0

0.003

0.268

0.1 M Tris, pH 7.5

–0.008

0.261

0.1 M Citrate, pH 5.0

0.015

0.249

10 mM EDTA

0.007

0.235a

1 M (NH4)2SO4

0.002

0.269

Data were obtained by mixing 5 µL of sample with 5 µL of the specified compound before adding 200 µL of dye reagent.

aMeasurements that differ from the control by more than 0.02 absorbance unit for blank values or more than 10% for the samples containing protein.

Data taken from ref. 7.

3. The dye does not bind to free arginine or lysine, or to peptides smaller than about 3000 Da (4,11). Many peptide hormones and other important bioactive peptides fall into the latter cat­egory, and the Bradford assay is not suitable for quantifying the amounts of such compounds.

4. The assay technique described here is subject to variation in sensitivity between indi­vidual proteins (see Table 2). Several modifications have been suggested that reduce this variability (5–7,12). Generally, these rely on increasing either the dye content or the pH of the solution. In one variation, adjusting the pH by adding NaOH to the reagent improves the sensitivity of the assay and greatly reduces the variation observed with different pro­teins (7). (This is presumably caused by an increase the proportion of free dye in the blue form, the ionic species that reacts with protein.) However, the optimum pH is critically dependent on the source and concentration of the dye (see Note 5). Moreover, the modi­fied assay is far more sensitive to interference from detergents in the sample.

Particular care should be taken when measuring the protein content of membrane frac­tions. The conventional assay consistently underestimates the amount of protein in mem-brane-rich samples. Pretreatment of the samples with membrane-disrupting agents such as NaOH or detergents may reduce this problem, but the results should be treated with caution (13). A useful alternative is to precipitate protein from the sample using calcium phosphate and remove contaminating lipids (and other interfering substances, see Note 1) by washing with 80% ethanol (9,10).

5. The amount of soluble dye in Coomassie Blue G250 varies considerably between sources, and suppliers’ figures for dye purity are not a reliable estimate of the Coomassie Blue G250 content (14). Generally, Serva Blue G is regarded to have the greatest dye content

Table 2 Comparison of the Response of Different Proteins in the Bradford Assay

Relative absorbance Protein Assay 1 Assay 2

Myelin basic protein Histone Cytochrome c Bovine serum albumin Insulin Transferrin Lysozyme α-Chymotrypsinogen Soybean trypsin inhibitor Ovalbumin γ-Globulin β-Lactoglobulin A Trypsin Aprotinin Gelatin Gramicidin S

139 130 128 100 89 82 73 55 52 49 48 20 18 13 — 5

— 175 142 100 — — — —

23 23 55 — 15 —

5 —

For each protein, the response is expressed relative to that of the same concentration of BSA. The data for assays 1 and 2 are recalculated from refs. 5 and 7, respectively. And should be used in the modified assays discussed in Note 4. However, the quality of the dye is not critical for routine protein determination using the method described in this chapter. The data presented in Fig. 1 were obtained using Coomassie Brilliant Blue G (C.I. 42655; product code B-0770, Sigma-Aldrich).

6. Whenever possible the protein used to construct the calibration curve should be the same as that being determined. Often this is impractical and the dye response of a sample is quantified relative to that of a “generic” protein. Bovine serum albumin (BSA) is com­monly used as the protein standard because it is inexpensive and readily available in a pure form. The major argument for using this protein is that it allows the results to be compared directly with those of the many previous studies that have used bovine serum albumin as a standard. However, it suffers from the disadvantage of exhibiting an unusu­ally large dye response in the Bradford assay, and thus, may underestimate the protein content of a sample. Increasingly, bovine γ-globulin is being promoted as a more suitable general standard, as the dye binding capacity of this protein is closer to the mean of those proteins that have been compared (Table 2). Because of the variation in response between different proteins, it is essential to specify the protein standard used when reporting mea­surements of protein amounts using the Bradford assay.

7. Generally, it is preferable to use a single new disposable polystyrene semimicrocuvette that is discarded after a series of absorbance measurements. Rinse the cuvette with reagent before use, zero the spectrophotometer on the reagent blank and then do not remove the cuvette from the machine. Replace the sample in the cuvette gently using a disposable polyethylene pipet.

8. The standard curve is nonlinear because of problems introduced by depletion of the amount of free dye. These problems can be avoided, and the linearity of the assay improved, by plotting the ratio of absorbances at 595 and 450 nm (15). If this approach is adopted, the absolute optical density of the free dye and dye–protein complex must be determined by measuring the absorbance of the mixture at each wavelength relative to that of a cuvette containing only water (and no dye reagent). As well as improving the linearity of the calibration curve, taking the ratio of the absorbances at the two wavelengths increases the accuracy and improves the sensitivity of the assay by up to 10-fold (15).

9. For routine measurement of the protein content of many samples the microassay may be adapted for use with a microplate reader (7,16). The total volume of the modified assay is limited to 210 µL by reducing the volume of each component. Ensure effective mixing of the assay components by pipetting up to 10 µL of the protein sample into each well before adding 200 µL of the dye reagent. If a wavelength of 595 nm cannot be selected on the microplate reader, absorbance may be measured at any wavelength between 570 nm and 610 nm. How­ever, absorbance measurements at wavelengths other than 595 nm will decrease the sensitiv­ity of response and may increase the minimum detection limit of the protocol.

10. For studies on the use of the Bradford assay in analyzing glycoproteins, see Note 9 in Chapter 3.

References

1. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248–254.

2. Chial, H. J., Thompson, H. B., and Splittgerber, A. G. (1993) A spectral study of the charge forms of Coomassie Blue G. Analyt. Biochem. 209, 258–266.

3. Compton, S. J. and Jones, C. G. (1985) Mechanism of dye response and interference in the Bradford protein assay. Analyt. Biochem. 151, 369–374.

4. Congdon, R. W., Muth, G. W., and Splittgerber, A. G. (1993) The binding interaction of Coomassie Blue with proteins. Analyt. Biochem. 213, 407–413.

5. Friendenauer, S. and Berlet, H. H. (1989) Sensitivity and variability of the Bradford pro­tein assay in the presence of detergents. Analyt. Biochem. 178, 263–268.

6. Reade, S. M. and Northcote, D. H. (1981) Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein. Analyt. Biochem. 116, 53–64.

7. Stoscheck, C. M. (1990) Increased uniformity in the response of the Coomassie Blue pro­tein assay to different proteins. Analyt. Biochem. 184, 111–116.

8. Spector, T. (1978) Refinement of the Coomassie Blue method of protein quantitation. A simple and linear spectrophotometric assay for less than 0.5 to 50 µg of protein. Analyt. Biochem. 86, 142–146.

9. Pande, S. V. and Murthy, M. S. R. (1994) A modified micro-Bradford procedure for elimi­nation of interference from sodium dodecyl sulfate, other detergents, and lipids. Analyt. Biochem. 220, 424–426.

10. Zuo, S.-S. and Lundahl, P. (2000) A micro-Bradford membrane protein assay. Analyt. Biochem. 284, 162–164.

11. Sedmak, J. J. and Grossberg, S. E. (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Analyt. Biochem. 79, 544–552.

12. Peterson, G. L. (1983) Coomassie blue dye binding protein quantitation method, in Meth­ods in Enzymology, vol. 91 (Hirs, C. H. W. and Timasheff, S. N., eds.), Academic Press, New York.

13. Kirazov, L. P., Venkov, L. G. and Kirazov, E. P. (1993) Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing frac­tions. Analyt. Biochem. 208, 44–48.

14. Wilson, C. M. (1979) Studies and critique of amido black 10B, Coomassie Blue R and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis. Analyt. Biochem. 96, 263–278.

15. Zor, T. and Selinger, Z. (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analyt. Biochem. 236, 302–308.

16. Redinbaugh, M. G. and Campbell, W. H. (1985) Adaptation of the dye-binding protein assay to microtiter plates. Analyt. Biochem. 147, 144–147.

From: The Protein Protocols Handbook, 2nd Edition Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ

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