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

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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 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 concentration range, the measured absorption at 540 nm is linear with respect to the concentration of total protein. This relationship allows a standard curve to be created that is used to calculate the concentration of an unknown sample. 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. 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. Key Words • Biuret Method • 8-Position Cell Changer • Total Protein • UV-Visible Spectroscopy Application Note: 51859 NH 2 Cu 2+ NH NH 2 NH 2 NH NH 2 Figure 1: Biuret reagent reacts with an alkaline solution of CuSO 4 to form a violet chelate compound Figure 3: Multi-Cell Method Set-up using VISIONcollect software 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 Figure 2: Experimental Method Set-up using VISIONcollect

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Page 1: The Biuret Method for the Determination of Total Protein Using an ...

The Biuret Method for the Determination ofTotal Protein Using an Evolution Array 8-Position Cell ChangerNicole Krueziger Keppy, Michael W. Allen, Ph.D, Thermo Fisher Scientific, Madison, WI, USA

Introduction

One commonly used method for determining the totalprotein in a sample is the Biuret method. The Biuret methodis 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 twopeptide bonds and produces a violet-colored chelateproduct which is measured by absorption spectroscopy at 540 nm. Over a givenconcentra tion range, the measured absorptionat 540 nm is linear with respect to theconcentration of totalprotein. This relationshipallows a standard curveto be created that is used to calculate theconcentration of anunknown sample.

Experiment and Results

The Biuret reagent was prepared by adding 3 g ofCuSO4•5H2O and 9 g of sodium potassium citrate to 500 mL of 0.2 N NaOH solution, followed by theaddition of 5 g of KI. The resulting solution was thenbrought 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 withsaline solution (8.5 g/L) according to Table 1. 3.0 mL ofBiuret reagent was added to each standard and sample,the solution was mixed well and incubated at roomtemperature for 30 minutes.

The standards and sample were analyzed using theBiuret method included in Thermo Scientific VISIONcollectsoftware with biological tests. To begin the measurement,select the Biuret Method from the provided method filesin Quantification Mode. The experimental method and 8-position cell changer set-up are shown in Figures 2 and 3 respectively.

Key Words

• Biuret Method

• 8-Position CellChanger

• Total Protein

• UV-VisibleSpectroscopy

ApplicationNote: 51859

NH2

Cu2+

NH

NH2

NH2

NH

NH2

Figure 1: Biuret reagent reacts with analkaline solution of CuSO4 to form a violetchelate compound

Figure 3: Multi-Cell Method Set-up using VISIONcollect software

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

Figure 2: Experimental Method Set-upusing VISIONcollect

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Standards 2 to 7 were measured at 540 nm usingstandard 1 as the reference sample, or blank. A linear fitwas applied to the standard results in Table 2 to obtainthe standard curve shown in Figure 4. The resultingcalibration curve exhibits a linear relationship with acorrelation coefficient (R2) of 0.9996. The unknownsample 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.

Conclusion

Automated quantitative analysis of protein is performedquickly and easily using the Thermo Scientific EvolutionArray UV-Visible spectrophotometer. The VISIONcollect™

software includes a pre-configured method for the Biuretassay, allowing further customization to individual laboratoryprotocols. Integration of the sample measurement and dataanalysis into VISIONcollect software saves time and improveslaboratory throughput by eliminating post-measurementdata manipulation. The 8-position cell changer enablesmeasurements to be taken without exchanging the cellsbetween measurements further increasing the efficiency of your methods.

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Figure 4: Biuret-Protein Complex Spectrum and Calibration Curve

Solution Concentration (µg/mL) Absorbance

Standard 2 200 0.0238Standard 3 400 0.0541Standard 4 700 0.0862Standard 5 1000 0.1304Standard 6 2000 0.2514Standard 7 3000 0.3817Unknown Sample 1553 0.1972

Table 2: Results of Protein Standards and the Sample using Biuret Method