Application Note: 51859
The Biuret Method for the Determination of Total Protein Using an Evolution Array 8-Position Cell Changer Nicole Krueziger Keppy, Michael W. Allen, Ph.D, Thermo Fisher Scientific, Madison, WI, USA
Introduction Key Words • Biuret Method • 8-Position Cell Changer • Total Protein • UV-Visible Spectroscopy
One commonly used method for determining the total protein in a sample is the Biuret method. The Biuret method is based on the complexation of Cu2+ to functional groups in the protein’s peptide bonds as shown in Figure 1. The formation of a Cu2+-protein complex requires two peptide bonds and produces a violet-colored chelate product which is measured by absorption spectroscopy at 540 nm. Over a given NH 2 NH 2 concentration range, the measured absorption at 540 nm is linear with respect to the NH NH concentration of total protein. This relationship Cu 2+ allows a standard curve to be created that is NH 2 NH 2 used to calculate the Figure 1: Biuret reagent reacts with an concentration of an alkaline solution of CuSO4 to form a violet unknown sample. chelate compound
Experiment and Results The Biuret reagent was prepared by adding 3 g of CuSO4• 5H2O and 9 g of sodium potassium citrate to 500 mL of 0.2 N NaOH solution, followed by the addition of 5 g of KI. The resulting solution was then brought to a total volume of 1 L with 0.2 N NaOH. Alternatively, the Biuret reagent is available from a variety of sources including Thermo Fisher Scientific. Protein standards and the sample were prepared with saline solution (8.5 g/L) according to Table 1. 3.0 mL of Biuret reagent was added to each standard and sample, the solution was mixed well and incubated at room temperature for 30 minutes.
The standards and sample were analyzed using the Biuret method included in Thermo Scientific VISIONcollect software with biological tests. To begin the measurement, select the Biuret Method from the provided method files in Quantification Mode. The experimental method and 8-position cell changer set-up are shown in Figures 2 and 3 respectively.
Figure 2: Experimental Method Set-up using VISIONcollect
Test Tube Number Reagent
3.0 mg/mL BSA (mL) Protein Sample (mL) Saline Solution (mL) Final Concentration (µg/mL)
1
2
3
4
5
6
7
8
–
0.2
0.4
0.7
1.0
2.0
3.0
–
–
–
–
–
–
–
–
1.0
3.0
2.8
2.6
2.3
2.0
1.0
–
2.0
0
200
400
700
1000 2000 3000
Table 1: Preparation of Protein Standards and the Sample
– Figure 3: Multi-Cell Method Set-up using VISIONcollect software
Standards 2 to 7 were measured at 540 nm using standard 1 as the reference sample, or blank. A linear fit was applied to the standard results in Table 2 to obtain the standard curve shown in Figure 4. The resulting calibration curve exhibits a linear relationship with a correlation coefficient (R2) of 0.9996. The unknown sample was measured in Quantification mode. Using the calibration curve the concentration of protein in the sample was calculated to be 1553 µg/mL, as shown in Table 2.
Solution
Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Unknown Sample
Concentration (µg/mL)
Absorbance
200 400 700 1000 2000 3000 1553
0.0238 0.0541 0.0862 0.1304 0.2514 0.3817 0.1972
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Table 2: Results of Protein Standards and the Sample using Biuret Method
Conclusion Automated quantitative analysis of protein is performed quickly and easily using the Thermo Scientific Evolution Array UV-Visible spectrophotometer. The VISIONcollect™ software includes a pre-configured method for the Biuret assay, allowing further customization to individual laboratory protocols. Integration of the sample measurement and data analysis into VISIONcollect software saves time and improves laboratory throughput by eliminating post-measurement data manipulation. The 8-position cell changer enables measurements to be taken without exchanging the cells between measurements further increasing the efficiency of your methods. Figure 4: Biuret-Protein Complex Spectrum and Calibration Curve
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