University:
Buffalo State University
Course:
CHE 111 | Fundamentals of Chemistry
Academic year:

2024
Views:

301

Pages:

2
Author:

ethan vicor

SEPARATION OF EGG WHITE PROTEINS BY SALTING OUT Basic theory. The addition of salt at low concentrations can increase solubility protein (Salting in), but the protein will experience salt precipitation on high concentration (Salting out). The salt commonly used is ammonium sulfate, sodium sulfate and sodium chloride. The precipitate formed can be dissolved back and the protein does not denature. The salting out mechanism is thought to occur through protein dehydration due to adding salt. Salt ions can attract water molecules that dissolve proteins until the protein becomes insoluble. In this experiment, separation will be carried out egg white protein by adding saturated ammonium sulfate salt. Test Tool : - Test tube - Stirring rod - Beaker - Centrifuge Material : - Ammonium sulfate salt - Protein solution (Egg White) - Tris buffer pH 7, 20 mM - Cellophane tube Procedure: A. Fractionation of egg white protein solution with the addition of (NH4)2SO4fed up (Salting Out Method) 1. Prepare five mL of egg white protein solution. 2. Add ammonium sulfate salt until it reaches 20% saturation (11.4 gr ammonium sulfate per 100 mL of solution) 3. Stir the mixture and leave for 5 minutes at room temperature. 4. Centrifuge for 15 minutes. 5. Transfer the supernatant into a new tube 6. The precipitate formed is called the 0 – 20% saturated ammonium sulfate fraction. 7. Dissolve the precipitate with 5 mL of Tris buffer pH 7. 8. Add ammonium sulfate salt to the supernatant from step 5 to reaches 50% saturation (18.9 grams of ammonium sulfate per 100 mL of solution). 9. Stir the mixture and leave for 5 minutes at room temperature. 10. Centrifuge for 15 minutes. 11. Transfer the supernatant into a new tube. 12. The precipitate formed is called the 20-50% saturated ammonium sulfate fraction. 13. Dissolve the precipitate with 5 mL of Tris buffer pH 7 14. Add ammonium sulfate salt to the supernatant from step 11 to reaches 70% saturation (13.7 grams of ammonium sulfate per 100 mL of solution). 15. Stir the mixture and leave for 5 minutes at room temperature 16. Centrifuge for 15 minutes. 17. Transfer the supernatant into a new tube. 18. The precipitate formed is called the 50 – 70% saturated ammonium sulfate fraction. 19. Dissolve the precipitate with 5 ml of Tris buffer pH 7. 20. Dialyze each ammonium sulfate fraction produc