| Contributor: |
The Laboratory of Michael Chamberlin at the University of California, Berkeley
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| This procedure for removing RNase from Bovine Serum Albumin using BioRex 70™ removes most, BUT NOT ALL, of RNase from Bovine Serum Albumin. To remove all of the RNase you should use a protocol that acetylates proteins. |
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A. Preparation of BioRex 70™ column
1. Wash the BioRex 70™ resin with 1 M NaOH for 3 hr (see Hint #1).
4. Rinse copiously with ddH2O until the eluate pH is 7.0.
5. Wash the BioRex 70™ resin with 1 M HCl for 3 hr (see Hint #1).
6. Rinse copiously with ddH2O until the eluate pH is 7.0.
7. Suspend BioRex 70™ resin in 1 M Sodium-Citrate Solution and incubate at room temperature for 1 hr.
8. Remove Sodium-Citrate Solution and add BioRex 70™ resin to column (Column should be 1.5 cm x 12 cm with a bed volume of approximately 21 ml).
9. Wash column containing BioRex 70™ with 100 mM Sodium-Citrate Solution (see Hint #2).
10. Make sure the column is equilibrated and the eluate pH is 5.75.
B. Removal of RNase from Bovine Serum Albumin
1. Dissolve 1.5 g Bovine Serum Albumin (BSA) in 100 mM Sodium-Citrate Buffer.
2. Mix until BSA is completely dissolved, then pH to 5.35 (pH is very important) with 1 M Citric Acid.
3. Wash the column with several volumes of 100 mM Sodium-Citrate Buffer before using.
4. Load sample onto the top of the column without disturbing the resin.
5. Allow column to flow.
6. Discard the first 10 ml.
7. Collect the remaining volume in a sterile flask.
8. Dialyze eluate against 2 liters of 2 mM Tris (include two additional volume changes over the course
of 24 hr).
9. Determine the final concentration of BSA (see protocol on Quantification of Protein Using the Bradford Assay) and check for RNase activity (see protocol on Quantification of RNase Activity).
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| 1 M NaOH |
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| 1 M Citric Acid |
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| 1 M Citric Acid |
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| 2 mM Tris, pH 8.0 |
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| 100 mM Sodium-Citrate Buffer |
| 100 mM Sodium Citrate
pH 5.35 using Citric Acid
Filter sterilize
pH is very important
A 1:10 dilution of 1 M Sodium-Citrate will not be a pH of 5.35 this is because the pH of Sodium-Citrate changes with the concentration of Sodium-Citrate
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| 1 M Sodium-Citrate Buffer |
| pH 5.35 using Citric Acid
pH is very important
1 M Sodium-Citrate
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| 1 M HCl |
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Sodium Hydroxide
Tris
Citric Acid
Sodium Citrate
Hydrochloric Acid
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1. This can easily be done in a beaker so long as you don't pour off a lot of the BioRex 70™ resin.
2. Washing the column will require at least 10 column volumes. Carefully monitor the eluate pH. The pH will initially rise and then return to 5.65.
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