Biochemical Techniques and Instrumentation

Procedure

1. Weigh 500mg of fish and grind well with a pestle and mortar using 5 to 10ml of the phosphate buffer, pH 7.0
2. Centrifuge and use the supernatant for protein estimation
3. Pipette out 0.1 and 0.2ml of the extract, in duplicate, into the test tubes and make up the volume to 1ml with distilled water
4. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0ml of protein working standard in a series of test tubes and make up the volume to 1ml with distilled water
5. A tube with 1ml of distilled water serves as the blank
6. Add 5ml of reagent C to all the tubes. Mix well and allow to stand for 10min.
7. Then, add 0.5ml of Folin–Ciocalteau reagent, mix well and incubate at room temperature in the dark for 30min
8. Read the blue color developed at 660nm in a spectrophotometer
9. Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis
10. From the graph, calculate the amount of protein in the sample