1) Standard Protein solution [200ug/ml].
2) Alkaline copper reagent.
3) Folin’s Ciocaltaeu reagent.
- Arrange the reagent solutions prepared, on the table.
- Label the test tubes with the volume taken and arrange them in a test tube rack .
- Pipette out the standard protein solution from the standard flask into the test tubes labelled [0.2ml-1ml].
- Pipette a known volume of the unknown solution to the tube labelled “unknown” arranged in the test tube rack.
- To the test tube labelled ‘Blank', add 1ml of distilled water using a micropipette.
- Volume in each test tube is made up to 1 ml by adding distilled water.
- Add 5ml of alkaline copper reagent to all the test tubes. Vortex and Incubate for 10 minutes at room temperature.
- The solution in all the test tubes has turned blue in colour.
- After incubation, add 600ul of Folin’s Ciocalteau reagent to all test tubes using micropipette. Vortex and Incubate for 20 minutes at room temperature.
- After incubation, the color intensity varies accordingly with the concentration of protein present in the tubes.
- Now record the absorbance of each solution at 650 nm using a colorimeter.
- Plot the absorbance against amount of protein in milligrams to get a standard calibration curve. Check the absorbance of unknown sample and determine the concentration of the unknown sample from the standard curve plotted.