Estimation of Protein by the FC-Method

Principle
Protein reacts with the Folin-Ciocalteu reagent (FC-reagent) to produce a colored complex. The color is due to the reaction of the alkaline copper sulfate with the protein and the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein. The intensity of color depends on the amount of these aromatic amino acids present in the protein, and will thus vary for DLF proteins.

Materials

1. Alkaline sodium carbonate solution:
   20 gm/liter sodium carbonate (Na₂CO₃) in 0.1 M/liter NaOH.
2. Copper sulfate: Sodium potassium tartrate solution 5 gm/liter-hydrated copper sulfate (CuSO₄-5H₂O) in 10 gm/liter sodium potassium tartrate prepare freshly by meaning stalk solution.
3. Alkaline solution: Prepare on the day of use by mixing 50 mL of 1 and 1 mL of 2.
4. FC-reagent: Dilute the commercial reagent with an equal volume of water on the day of use. This is a solution of sodium tangstate and sodium molybdate in phosphoric acid and HCl.
5. 5. Standard protein solution: Weigh 100 mg egg albumin and dissolve it in approximately 50 mL of water. Make up the volume to 100 mL.
6. Working protein solution: Pipette out 20 mL of stock protein solution into another 100 mL volumetric flask and make up the volume to 100 mL.

Procedure
Pipette out protein solution in the range of 0 to 200 mg/1 mL into d/f test tubes. Make up the volume of all the test tubes to 1 mL with water. Add 5 mL of alkaline solution to each test tube, mix thoroughly, and allow to stand at room temperature for 10 minutes. Add 0.5 mL of dilute FC-reagent rapidly with immediate mixing. After 30 minutes read the extinction against the appropriate blank at 650 nm.

Estimate the protein concentrate of the unknown solution after preparing a standard curve.

Result:
The given unknown solution contains .............. mg/mL of protein.