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

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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, USAIntroductionOne 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 proteins 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 ResultsThe Biuret reagent was prepared by adding 3 g ofCuSO45H2O 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-VisibleSpectroscopyApplicationNote: 51859NH2Cu2+NHNH2NH2NHNH2Figure 1: Biuret reagent reacts with analkaline solution of CuSO4 to form a violetchelate compoundFigure 3: Multi-Cell Method Set-up using VISIONcollect softwareTest Tube NumberReagent 1 2 3 4 5 6 7 83.0 mg/mL BSA (mL) 0.2 0.4 0.7 1.0 2.0 3.0 Protein Sample (mL) 1.0Saline Solution (mL)3.0 2.8 2.6 2.3 2.0 1.0 2.0Final Concentration 0 200 400 700 1000 2000 3000 (g/mL)Table 1: Preparation of Protein Standards and the SampleFigure 2: Experimental Method Set-upusing VISIONcollectStandards 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 g/mL, as shown in Table 2.ConclusionAutomated quantitative analysis of protein is performedquickly and easily using the Thermo Scientific EvolutionArray UV-Visible spectrophotometer. The VISIONcollectsoftware 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.Part of Thermo Fisher ScientificIn addition to these offices, Thermo FisherScientific maintains a network of represen -tative organizations throughout the world.Africa-Other+27 11 570 1840Australia+61 2 8844 9500Austria+43 1 333 50 34 0Belgium+32 2 482 30 30Canada+1 800 530 8447China+86 10 8419 3588Denmark+45 70 23 62 60 Europe-Other+43 1 333 50 34 0Finland / Norway /Sweden+46 8 556 468 00France+33 1 60 92 48 00Germany+49 6103 408 1014India+91 22 6742 9434Italy+39 02 950 591Japan +81 45 453 9100Latin America+1 608 276 5659Middle East+43 1 333 50 34 0Netherlands+31 76 579 55 55South Africa+27 11 570 1840Spain+34 914 845 965Switzerland+41 61 716 77 00UK+44 1442 233555USA+1 800 532 4752www.thermo.comAN51859_E 12/09MThermo Electron ScientificInstruments LLC, Madison, WIUSA is ISO Certified.2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.Figure 4: Biuret-Protein Complex Spectrum and Calibration CurveSolution Concentration (g/mL) AbsorbanceStandard 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 0.1972Table 2: Results of Protein Standards and the Sample using Biuret Method


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