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Construction of Protein Standard Curve using Folin’s Lowry Method
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Materials Required:


1)    Standard Protein solution [200ug/ml].


2)    Alkaline copper reagent.


3)    Folin’s Ciocaltaeu reagent.


4)    Colorimeter


5)    Pipettes.

 

Working Steps:

 

  1. Arrange the reagent solutions prepared, on the table.
  2. Label the test tubes with the volume taken and arrange them  in a test tube rack .
  3. Pipette out the standard protein solution from the standard flask into the test tubes labelled [0.2ml-1ml].
  4. Pipette a known volume of the   unknown solution to the tube labelled “unknown” arranged in the  test tube rack.
  5. To the test tube labelled  ‘Blank', add 1ml of distilled water using a micropipette.
  6. Volume in each test tube is  made up to 1 ml by adding distilled water.
  7.  Add 5ml of alkaline copper reagent to all the test tubes.  Vortex and  Incubate for 10 minutes at room temperature.
  8. The solution in all the test tubes has turned blue in colour.
  9.  After incubation, add 600ul of Folin’s Ciocalteau reagent to all test tubes using micropipette. Vortex and  Incubate for 20 minutes at room temperature.
  10. After incubation, the color intensity varies accordingly with the concentration of protein present in the tubes.
  11. Now record the absorbance of each solution at 650 nm using a colorimeter.
  12. 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.
     

 

 

 

 

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