Determine Procedure for determination of protein content?
Now carry out the procedure as enumerated herewith:
1. Preparation of standard albumin: A standard solution of 2 mg albumin/ml is prepared in distilled water. For this weigh 200 mg of albumin and transfer This amount into a 100 ml volumetric flask. Make up the volume to the mark with distilled water.
2. Colour development for standard solution:
- Take different volumes of standard albumin solution (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml) in different test tubes
- In each case, make up the volume to 3 ml with distilled water.
- Add 3 ml of biuret reagent to each test tube and keep the tubes at room temperature for 30 minutes for colour development after shaking.
3. Preparation of unknown solution: Take the unknown solution and dilute the given solution to 100 ml mark and take one volume (e.g. 2.0 ml) in duplicate similar to that prepared for the standard. Add all other reagents just as you did for the standard solution.
4. Preparation of blank: Pipet 3 ml of distilled water. (Do not add the standard solution in the blank). Now repeat the above procedure for colour development.
5. Measurement of O.D: Read the absorbance of all solutions at 540 nm after setting the colorimeter to 100% transmittance with the blank solution.
6. Standard Curve: Plot a graph between concentration albumin in standard solution and O.D.
7. Calculate the amount of protein with the help of the standard curve.