METHOD 3 This method (commonly referred to as the Bradford assay) is based on the absorption shift from 470 nm to 595 nm observed when the acid blue 90 dye binds to protein. The acid blue 90 dye binds most readily to arginine and lysine residues in the protein which can lead to variation in the response of the assay to different proteins. The protein used as reference substance must therefore be the same as the protein to be determined. There are relatively few interfering substances, but it is preferable to avoid detergents and ampholytes in the test sample. Highly alkaline samples may interfere with the acidic reagent. Use distilled water R to prepare all buffers and reagents used for this method. Test solution. Dissolve a suitable quantity of the substance to be examined in the prescribed buffer to obtain a solution having a concentration within the range of the standard curve. Reference solutions. Dissolve the reference substance for the protein to be determined in the prescribed buffer. Dilute portions of this solution with the same buffer to obtain not fewer than five reference solutions having protein concentrations evenly spaced over a suitable range situated between 0.1 mg/ml and 1 mg/ml. Blank. Use the buffer used to prepare the test solution and the reference solutions. Acid blue 90 reagent. Dissolve 0.10 g of acid blue 90 R in 50 ml of alcohol R. Add 100 ml of phosphoric acid R, dilute to 1000 ml with distilled water R and mix. Filter the solution and store in an amber bottle at room temperature. Slow precipitation of the dye occurs during storage. Filter the reagent before using. Procedure. Add 5 ml of acid blue 90 reagent to 0.100 ml of each reference solution, of the test solution and of the blank. Mix by inversion. Avoid foaming, which will lead to poor reproducibility. Determine the absorbances (2.2.25) of the standard solutions and of the test solution at 595 nm, using the blank as compensation liquid. Do not use quartz (silica) spectrophotometer cells because the dye binds to this material. Calculations. The relationship of absorbance to protein concentration is non-linear; however, if the range of concentrations used to prepare the standard curve is sufficiently small, the latter will approach linearity. Plot the absorbances of the reference solutions against protein concentrations and use linear regression to establish the standard curve. From the standard curve and the absorbance of the test solution, determine the concentration of protein in the test solution.
METHOD 4 This method (commonly referred to as the bicinchoninic acid or BCA assay) is based on reduction of the cupric (Cu2+) ion to cuprous (Cu1+) ion by protein. The bicinchoninic acid reagent is used to detect the cuprous ion. Few substances interfere with the reaction. When interfering substances are present their effect may be minimised by dilution, provided that the concentration of the protein to be determined remains sufficient for accurate measurement. Alternatively, the protein precipitation procedure given in Method 2 may be used to remove interfering substances. Because different protein species may give different colour response intensities, the reference protein and protein to be determined must be the same. Use distilled water R to prepare all buffers and reagents used for this method. Test solution. Dissolve a suitable quantity of the substance to be examined in the prescribed buffer to obtain a solution having a concentration within the range of the concentrations of the reference solutions. Reference solutions. Dissolve the reference substance for the protein to be determined in the prescribed buffer. Dilute portions of this solution with the same buffer to obtain not fewer than five reference solutions having protein concentrations evenly spaced over a suitable range situated between 10 μg/ml and 1200 μg/ml. Blank. Use the buffer used to prepare the test solution and the reference solutions. BCA reagent. Dissolve 10 g of disodium bicinchoninate R, 20 g of sodium carbonate monohydrate R, 1.6 g of sodium tartrate R, 4 g of sodium hydroxide R, and 9.5 g of sodium hydrogen carbonate R in distilled water R. Adjust, if necessary, to pH 11.25 with a solution of sodium hydroxide R or a solution of sodium hydrogen carbonate R. Dilute to 1000 ml with distilled water R and mix. Copper-BCA reagent. Mix 1 ml of a 40 g/l solution of copper sulphate R and 50 ml of BCA reagent. Procedure. Mix 0.1 ml of each reference solution, of the test solution and of the blank with 2 ml of the copper-BCA reagent. Incubate the solutions at 37 °C for 30 min, note the time and allow the mixtures to cool to room temperature. Within 60 min of the end of incubation, determine the absorbances (2.2.25) of the reference solutions and of the test solution in quartz cells at 562 nm, using the blank as compensation liquid. After the solutions have cooled to room temperature, the colour intensity continues to increase gradually. Calculations. The relationship of absorbance to protein concentration is non-linear ; however, if the range of concentrations used to prepare the standard curve is sufficiently small, the latter will approach linearity. Plot the absorbances of the reference solutions against protein concentrations and use linear regression to establish the standard curve. From the standard curve and the absorbance of the test solution, determine the concentration of protein in the test solution.
METHOD 5 This method (commonly referred to as the biuret assay) is based on the interaction of cupric (Cu2+) ion with protein in alkaline solution and resultant development of absorbance at 545 nm. This test shows minimal difference between equivalent IgG and albumin samples. Addition of the sodium hydroxide and the biuret reagent as a combined reagent, insufficient mixing after the addition of the sodium hydroxide, or an extended time between the addition of the sodium hydroxide solution and the addition of the biuret reagent will give IgG samples a higher response than albumin samples. The trichloroacetic acid method used to minimise the effects of interfering substances also can be used to determine the protein content in test samples at concentrations below 500 μg/ml. Use distilled water R to prepare all buffers and reagents used for this method. Test solution. Dissolve a suitable quantity of the substance to be examined in a 9 g/l solution of sodium chloride R to obtain a solution having a concentration within the range of the concentrations of the reference solutions. Reference solutions. Dissolve the reference substance for the protein to be determined in a 9 g/l solution of sodium chloride R. Dilute portions of this solution with a 9 g/l solution of sodium chloride R to obtain not fewer than three reference solutions having protein concentrations evenly spaced over a suitable range situated between 0.5 mg/ml and 10 mg/ml. Blank. Use a 9 g/l solution of sodium chloride R. Biuret reagent. Dissolve 3.46 g of copper sulphate R in 10 ml of hot distilled water R, and allow to cool (Solution A). Dissolve 34.6 g of sodium citrate R and 20.0 g of anhydrous sodium carbonate R in 80 ml of hot distilled water R, and allow to cool (Solution B). Mix solutions A and B and dilute to 200 ml with distilled water R. Use within 6 months. Do not use the reagent if it develops turbidity or contains any precipitate. Procedure. To one volume of the test solution add an equal volume of a 60 g/l solution of sodium hydroxide R and mix. Immediately add biuret reagent equivalent to 0.4 volumes of the test solution and mix rapidly. Allow to stand at a temperature between 15 °C and 25 °C for not less than 15 min. Within 90 min of addition of the biuret reagent, determine the absorbances (2.2.25) of the reference solutions and of the test solution at the maximum at 545 nm, using the blank as compensation liquid. Any solution that develops turbidity or a precipitate is not acceptable for calculation of protein concentration. Calculations. The relationship of absorbance to protein concentration is approximately linear within the indicated range of protein concentrations for the reference solutions. Plot the absorbances of the reference solutions against protein concentrations and use linear regression to establish the standard curve. Calculate the correlation coefficient for the standard curve. A suitable system is one that yields a line having a correlation coefficient not less than 0.99. From the standard curve and the absorbance of the test solution, determine the concentration of protein in the test solution. Interfering substances. To minimise the effect of interfering substances, the protein can be precipitated from the test sample as follows : add 0.1 volumes of a 500 g/l solution of trichloroacetic acid R to 1 volume of a solution of the test sample, withdraw the supernatant layer and dissolve the precipitate in a small volume of 0.5 M sodium hydroxide. Use the solution obtained to prepare the test solution.
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