Biuret test for protein

Biuret reagent reacts with peptide bonds, turning from blue to purple. Unlike the Bradford test it will give equally good results with any protein, but it is unable to detect the low concentrations that can be achieved with the Bradford test.

A qualitative test can be performed simply by adding equal quantities of 1% sodium (or potassium) hydroxide and a 1% solution of copper sulphate to the sample. If the solution turns purple it means that protein is present.

The results described here use quantitative Biuret solution which will give a good estimate of protein concentrations in the range 0.1-10 mg/cm3.

Safety

eye-protection
gloves
Biuret reagent contains 0.75M sodium hydroxide.
THE USE OF EYE PROTECTION MUST BE STRICTLY ENFORCED
In the event of eye contact flood the eye gently with running water for ten minutes and seek medical attention.
corrosive
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Method

Biuret reagent

    Solution 1
    Copper sulphate.5H2O
    Sodium potassium tartrate
    0.75g
    0.3g
    Dissolve in 250cm3 H2O

    Solution 2
    Sodium hydroxide 15g
    Dissolve in 150cm3 H2O

    Add 2 to 1 mixing thoroughly and make up to 500cm3.
    If a precipitate forms 0.5g potassium iodide can be added

    Label the stock solution 'CORROSIVE' corrosive

  • To perform the quantitative test add 2 cm3 of Biuret reagent to 0.5 cm3 of sample.
  • Allow the mixture to stand for 10 minutes then read absorbance in green light, (~565nm).

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Results

Similar results were obtained using either powdered egg white or gelatine as the protein standard

    Biuret graph

pdf Click to download a pdf file containing this information.

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