| Contributor: |
The Laboratory of Michael Chamberlin at the University of California, Berkeley
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| Acetylation will completely destroy RNase activity. However acetic anhydride treatment of Bovine Serum Albumin (BSA) can result in problems with specific applications. Therefore, it is very important that you verify that the acetic anhydride, RNase free BSA will work in your specific experiments before preparing a large batch. |
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1. Add 2 g of Bovine Serum Albumin (BSA) to 100 ml of 50 mM NaHCO3.
2. Place the BSA solution on a stir plate with constant gentle stirring.
3. Place a pH probe in the solution and monitor the pH. Keep the pH at 8.0 by adding drop wise 1 M Na2HCO3.
4. Remove 600 μl of Acetic Anhydride from stock and place this in a beaker.
5. SLOWLY add Acetic Anhydride to the BSA solution. Do this drop wise by transferring a small amount of Acetic Anhydride into the BSA solution via a Pasteur pipette (see Hint #1).
6. Keep the pH at 8.0 by adding drop wise 1 M Na2HCO3.
7. After adding the Acetic Anhydride Solution to the BSA double check the pH and make sure that it is stable at 8.0.
8. Dialyze overnight the BSA solution against 2 liters of Sterile ddH2O (see protocol on Dialysis of Proteins).
9. Replace the ddH2O with another 2 liters of Sterile ddH2O and dialyze again overnight.
10. Determine the final protein concentration by a method that stains protein (see protocol on Quantification of Protein Concentration) since anhydride treated BSA does not have an energy maximum at 280 nanometers.
11. Check for RNase activity.
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| Sterile ddH2O
Sterilize 4 liters of ddH2O
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| 1 M Sodium Carbonate (Na2HCO3) |
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| 1 M NaHCO3 |
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| 50 mM Sodium Bicarbonate (NaHCO3) |
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Bovine Serum Albumin (BSA)
Acetic Anhydride
Sodium Carbonate
Sodium Bicarbonate
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1. The process of adding 600 μl of Acetic Anhydride to the BSA solution should take approximately 30 min.
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