Measurement of protein solutions concentrations by biuret protein assay

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Title: Measurement of protein solutions concentrations by biuret protein assay


To study the principle a protein assay.

To determine the absorbance rate of protein at different concentration by using             spectrophotometer.

To determine the concentration of unknown sample from the graph.


Protein quantitation is often necessary before processing protein samples for isolation, separation and analysis by chromatographic, electrophoresis and immunochemical techniques. Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration.        

Instead, several colorimetric, reagent-based protein assay techniques have been developed that are used by nearly every laboratory involved in protein research. Protein is added to the reagent, producing a colour change in proportion to the amount added. Proteins concentration is determined by reference to a standard curve consisting of known concentrations of a purified reference protein.

Most pure protein solutions containing 1 mg/mL of protein have an absorbance of about 1.0 when the light path is 1 cm. This method is simple, rapid, and allows for full recovery of the protein. However, many other biochemicals absorb near this wavelength, making an accurate quantitation difficult. Furthermore, different proteins absorb to different extents depending upon their aromatic amino acid content. To avoid these problems, the Biuret test was developed. The Biuret reagent is made of sodium hydroxide and copper sulphate. The copper atoms of Biuret solution (CuSO4 and KOH) will react with peptide bonds, producing colour change. The blue reagent turns violet in the presence of proteins, and changes to pink when combined with short-chain polypeptides.

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Bovine serum albumin (BSA)

Biuret reagent


Test tubes




  1. A Biuret reagent, consisting of 2.25 gm sodium potassium tartrate, 0.75 gm Copper sulphate x 5 H2O, 1.25 gm potassium iodide was prepared, all dissolved in order in 100 mL 0.2 M NaOH (0.8 gm/100 mL). The volume was brought to 250 mL with distilled water. If a black precipitate forms, then it was discarded.
  2. A serial dilution of 2, 4, 6, 8, and 10 mg/mL was prepared by using a stock solution of bovine serum albumin (10 mg/mL). (Final volume ...

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***** A thorough consideration of the use of the biuret test for protein assays. Good use of A level scientific language throughout.