| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| This protocol uses harsher conditions to purify the protein from Baculovirus-infected insect cells than those used in the Protocol for Protein Purification of Py-Tagged Proteins from Vaccinia Virus-Infected Cells Using Alpha-PY-Protein-G-Sepharose. If you do not want to elute the protein off the column with the peptide, perform high pH elution (see Protein Purification of Py-Tagged Proteins from Vaccinia Virus-Infected Cells Using Anti-Py-Protein-G-Sepharose). |
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1. Prepare Anti-Py Antibody-Protein G Sepharose complex by binding anti-Py monoclonal antibodies to Protein G Sepharose followed by Dimethylpimelimidate crosslinking (see Protocol on Preparation of Antibody-Protein G Sepharose Beads). Store the Sepharose at 4°C.
2. For Baculovirus-infected cells, thaw frozen cell pellets in 5 volumes of Buffer R at room temperature.
3. Centrifuge the cell suspension for 10 min at 12,000 X g.
4. Add NaCl to the sample to a final concentration of 80 mM, and add N-Octylglucoside to 0.1% (see Hint #1).
5. Incubate the sample for 10 min at room temperature.
6. Centrifuge the sample for 10 min at 12,000 X g and recover the supernatant.
7. Prepare a 0.2 ml or larger α-Py-Protein-G-Sepharose column, depending on the capacity of the column material and the concentration of the tagged protein in your preparation (this requires empirical optimization). Rinse the column with 2 volumes of Wash Buffer 1.
5. Load the extract supernatant, collect the flow through, and reapply to the column at a slow enough flow rate until approximately 30 min has elapsed at room temperature. Continue to re-apply until at least this amount of time has elapsed (see Hint 2).
6. Wash the column with 10 to 20 column volumes (CV) of Wash Buffer 2.
7. Elute and collect the bound protein from the column with 3 CV of Elution Buffer at a rate of between 4 to 10 CV per hr.
8. The column can be recycled by washing it with 5 volumes of Carbonate Buffer (see Hint #3 and #4).
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| PBS |
| 1.8 mM NaH2HPO4
pH 7.2
4.3 mM Na2HPO4
2.7 mM KCl
137 mM NaCl
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| Wash Buffer 2 |
| Make up in PBS
0.5% (v/v) Igepal CA-630
2 mM 2-Mercaptoethanol
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| Wash Buffer 1 |
| 100 mM NaCl
10 μg/ml Leupeptin
4 mM 2-Mercaptoethanol
1 mM MgCl2
1 mM EGTA
20 mM Tris-HCl, pH 8.2
0.1% (v/v) N-Octylglucoside
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| Buffer R |
| 10 μg/ml Leupeptin
4 mM 2-Mercaptoethanol
1 mM MgCl2
1 mM EGTA
20 mM Tris-HCl, pH 8.2
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| Carbonate Buffer |
| 100 mM Na2CO3, pH 10.5
0.5% (v/v) Igepal CA-630
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| Py Peptide Stock |
| Keep a working stock at 4°C to reduce freeze thaw cycles
25 mg/ml peptide (EYMPME)
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| Elution Buffer |
| 0.1% (v/v) N-Octylglucoside (optional)
25 μg/ml Py Peptide from Py Peptide Stock
Make up in PBS.
2 mM 2-Mercaptoethanol
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Leupeptin
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
IGEPAL CA-630
Sodium Chloride
Magnesium Chloride
Sodium Carbonate
EGTA
Tris
Dimethylpimelimidate
Anti Py Antibody-Protein-G Sepharose
Peptide (EYMPME)
N-Octylglucoside
2-Mercaptoethanol
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1. For other types of cells or extracts prepared in different ways, try to get the sample in a buffer as similar to Buffer R/80 mM NaCl/0.1% (v/v) N-Octylglucoside as possible by dilution or dialysis and/or the addition of compounds.
2. Batch absorption can also be used, especially for smaller volumes. In that case, rock for 1 hr at room temperature instead of passing the extract over a column.
3. The column can be reused about 20 times for the same protein. If there is more than 0.1 mM DTT present in the extracts and/or if the elution periods are much longer than 3 hr, do not use the column more than 3 to 5 times.
4. In some cases, the high concentration of negatively charged peptide present in the Elution Buffer can interfere with the activity of your protein. To clear the eluate with (most of) the peptide, dialyze the eluate in a dialysis membrane with a cut off of MW 10,000 Da or higher against a protein buffer of choice containing high salt (0.8 to 1 M NaCl). Follow with a lower salt dialysis buffer if necessary. You can also run additional columns.
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