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BACKGROUND
Biuret reagent can be used for estimating the amount of protein due to the formation of peptide bonds. Biuret reagent contains sodium hydroxide and hydrated copper sulfate along with potassium sodium tartarate that stabilizes the cupric ions after reacting with copper sulfate. These cupric ions are responsible for dislocating the peptide hydrogen atoms under alkaline conditions.1
The main objective of this experiment is to estimate the total proteins present in plasma by biuret method.
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REQUIREMENTS
Chemicals: A protein standard 5 mg BSA/ml
Plasma sample
Biuret reagent
Others: Spectrophotometer or colorimeter
Test tubes
Pipette
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PROCEDURE
Preparation of Biuret reagent
Add 3 g of copper sulphate and 9 g of sodium potassium tartarate in 500 ml sodium hydroxide; then dissolve 5 g of potassium iodide to make it 1 litre.
Test procedure
Label the test tubes and in each test tubes accordingly. Add working standard of quantity 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1 ml. Then take another test tube and put 1 ml of the sample to be tested in it. Take from this and add to working standard test tube to make it 1 ml approx. Take distilled water in a separate test tube to treat it as blank. Now add about 3 ml of Biuret reagent to all the test tubes. Vortex all the samples to properly mix all the contents in the test tubes and warm it at a temperature of 37oC for about 20 minutes. Now keep all the content to cool down and measure the absorbance at 540 nm against that of blank. A standard curve now should be plotted by taking protein concentration in X-axis and absorbance in Y-axis. This curve is used for estimating the total amount of protein in the given sample.2
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CONCLUSION
This is one of the simplest method for calculating the amount of plasma protein. The amount of protein is calculated in mg protein/ml.
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REFERENCES
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Gornall AG, Bardawill CJ, David MM. J Biol Chem. 1949;177:751-66.
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Ryan MT Chopra RK. Biochim Biophys Acta. 1976;427:337-49.
