1. Weighing balance
2. Spirulina tablets
3. Watch glass
5. 100 ml beaker
6. Sonication chamber
7. Ammonium Sulphate powder
8. Magnetic stirrer
- Weigh 2g of spirulina powder in a TARED weigh balance and transfer it into a 100ml beaker containing 0.1M potassium phosphate buffer at pH=7. Stir the contents well.
- Sonicate the spirulina solution in a sonicator for 1 minute at intervals of 5 minutes for 5-6 times to break open the cells and to release the proteins into the solution.
- Centrifuge the suspension in a cooling centrifuge at 24000rpm for 20 minutes at 40C.
- The supernatant is collected into a beaker and the volume of the solution is made up to 100ml by adding 0.1M potassium phosphate buffer.
- Add 29.5g solid ammonium sulfate into the supernatant solution to precipitate the phycobiliproteins. Stir well for 15 minutes.
- Centrifuge the solution in a cooling centrifuge at 16000rpm for 10minutes at 40C.
- The supernatant is discarded and the protein pellet is then resuspended in 25ml 0.1M phosphate buffer at pH=7.This resuspended protein solution is transferred to conical flask for further studies.
Protein Denaturation Studies:
- Transfer 3mL of 0.1M Potassium Phosphate buffer into a cuvette using a pipette, to be used as the blank.
- Add 3 ml of Potassium Phosphate buffer using a pipette to another cuvette.
- Add 100 µl protein solutions from the conical flask into this cuvette and mix it well using the pipette.
- These cuvettes containing the sample and the blank are inserted into the sample holders of a UV-Vis spectrometer whose base line correction is already done using 0.1M Potassium phosphate buffer. The UV spectrum generated at 625nm is recorded.
- The solution is discarded and the cuvette is washed and dried.
- Now 1.5 ml 0.1M potassium phosphate buffer is taken in the cuvette.
- Add 1.50 ml of the denaturant, 8M potassium thiocyanate, to the cuvette.
- Add 100 µl protein solution and mix well using a pipette.
- The cuvette containing the sample is inserted into the sample holder of UV-Vis spectrometer against the Blank solution . The UV spectrum generated at 625nm is recorded.
- The solution is discarded and the cuvette is washed well and dried.
- Transfer 2.25ml of 0.1M potassium phosphate buffer in the cuvette.
- Add 750ul of the second denaturant, 8M urea, to the cuvette.
- Add 100 ul of protein solution to this cuvette. Mix well using a pipette.
- The cuvette containing the sample is inserted into the sample holder of UV-Vis spectrometer against the Blank solution. The UV spectrum generated at 625nm is recorded.
- Remove the cuvettes from the UV spectrometer and wash them properly.
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