Aim- Prepare standard curve by Biuret test for Quantitative estimation of protein.

Theory:- The protein are made up of amino acids containing peptide bonds. Two or more
peptide bond react with copper sulphate and result in violet colored product. The Cu atom from
complex with four of nitrogen atoms of peptide chain and develop the color. The number of
peptide bonds present in a sample governs the color intensity of the sample.
Requirements-
Unknown Protein solution, Biuret reagent‫٭‬, test tubes
‫٭‬Biuret reagent- Dissolve 3gm CuSO4.5H2O and %gm of sodium potassium tartrate in 500 ml of
0.2N of NaOH solution. Add 5gm of potassium iodide and make the volume to 1lit by adding
0.2N NaOH solution.
Procedure-
1- Suspend 1.5ml of protein solution in a test tube containing 1N NaOH solution at 100ºC for 4-5
minutes.
2- Add 3ml of Biuret reagent in this mixture.
3- Mix the content and incubate in the water bath set at 37ºC for 10 minutes.
4- Cool the test tubes by keeping it to room temperature.
5- Measure the OD at 540nm in a colorimeter.
Result;- Plot a graph between protein concentration Vs optical density.