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Structural Studies of Phycobiliproteins from Spirulina


Flash content link: https://vlab.amrita.edu/repo/BIOTECH/BIC/Structural_Studies_of_Phycobiliproteins_from_Spirulina/index.swf


Materials Required


1.    Weighing balance
2.    Spirulina tablets
3.    Watch glass
4.    Spatula.
5.    100 ml beaker
6.    Sonication chamber
7.    Ammonium Sulphate powder
8.    Magnetic stirrer



Working Steps:

  1. 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.
  2. 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.
  3. Centrifuge the suspension in a cooling centrifuge at 24000rpm for 20 minutes at 40C.
  4. 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.
  5. Add 29.5g solid ammonium sulfate into the supernatant solution to precipitate the phycobiliproteins. Stir well for 15 minutes.
  6. Centrifuge the solution in a cooling centrifuge   at 16000rpm for 10minutes at 40C. 
  7. 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:


  1. Transfer 3mL of 0.1M Potassium Phosphate buffer into a cuvette using a pipette, to be used as the blank.
  2. Add 3 ml of Potassium Phosphate buffer using a pipette to another cuvette.
  3. Add 100 µl protein solutions from the conical flask into this cuvette and mix it well using the pipette.
  4. 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.
  5. The solution is discarded and the cuvette is washed and dried.
  6. Now 1.5 ml 0.1M potassium phosphate buffer is taken in the cuvette.
  7. Add 1.50 ml of the denaturant, 8M potassium thiocyanate, to the cuvette.
  8. Add 100 µl protein solution and mix well using a pipette.
  9. 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.
  10. The solution is discarded and the cuvette is washed well and dried.
  11. Transfer   2.25ml of 0.1M potassium phosphate buffer in the cuvette.
  12. Add 750ul of the second denaturant, 8M urea, to the cuvette.
  13. Add 100 ul of protein solution to this cuvette. Mix well using a pipette.
  14. 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.
  15. Remove the cuvettes from the UV spectrometer and wash them properly.





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