The Biuret Method for the Determination of Total Protein Using an ...

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
Key Words
• Biuret Method
• 8-Position Cell
Changer
• Total Protein
• UV-Visible
Spectroscopy
Introduction
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 Cu 2+ to functional groups
in the protein’s peptide bonds as shown in Figure 1.
The formation of a Cu 2+ -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
concentra tion range,
NH 2 NH 2
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
concentration of an Figure 1: Biuret reagent reacts with an
alkaline solution of CuSO
unknown sample.
4 to form a violet
chelate compound
Experiment and Results
The Biuret reagent was prepared by adding 3 g of
CuSO 4
•5H 2 O 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.
Test Tube Number
Reagent 1 2 3 4 5 6 7 8
3.0 mg/mL
BSA (mL)
– 0.2 0.4 0.7 1.0 2.0 3.0 –
Protein
Sample (mL)
– – – – – – – 1.0
Saline
Solution (mL)
3.0 2.8 2.6 2.3 2.0 1.0 – 2.0
Final
Concentration 0 200 400 700 1000 2000 3000 –
(µg/mL)
Table 1: Preparation of Protein Standards and the Sample
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
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 (R 2 ) 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.
Figure 4: Biuret-Protein Complex Spectrum and Calibration Curve
Solution Concentration (µg/mL) Absorbance
Standard 2 200 0.0238
Standard 3 400 0.0541
Standard 4 700 0.0862
Standard 5 1000 0.1304
Standard 6 2000 0.2514
Standard 7 3000 0.3817
Unknown Sample 1553 0.1972
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.
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