1. Extraction of protein from sample

• Weigh 5 g of the given sample and grind well with pestle and mortar with 10 ml of distilled water.
• Centrifuge at 5,000 rpm and make up volume of the supernatant to 100ml with distilled water for the protein estimation.

2. Estimation of protein

• Pipette out 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard into a series of test tubes.
• Pipette out 1ml distilled water into another test tube as blank.
• Pipette out 0.1 and 0.2ml of the sample extract into 2 more test tubes as samples.
• Make up the volume to 1ml in all the test tubes with distilled water.
• Add 5ml of reagent C.
• Mix well and allow standing for 10min.
• Then add 0.5 ml of reagent D.
• Mix well and allow standing at room temperature in dark for 30 min.
• Measure the absorption of the colour developed at 660 nm in a spectrophotometer.
• Draw a graph with concentration of BSA along the X axis and absorbance on the Y axis and calculate the amount of protein present in the given sample.