| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
|
| |
| This protocol was developed to purify Py-tagged proteins from Vacinia virus-infected HeLa cell extracts or L2 cell lysates. It works very well using mild conditions for the protein and is performed at 4°C. |
| |
1. Prepare the anti-Py-Protein Antibody-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). After conjugation, store the prepared Sepharose at 4°C (see Hint #1).
2. Centrifuge cellular extract containing the Py-fusion protein for 10 min in a microcentrifuge at full speed at 4°C.
3. Wash an appropriate volume of Sepharose beads twice, very briefly, with Py-Wash Buffer, preferably without DTT (see Hint #2).
4. Incubate the beads with the cellular extract supernatant from Step #2 with rotation for 3 hr at 4°C (see Hint #3).
5. Wash the Sepharose resin 4 times with at least 20 bead volumes of Py Wash Buffer for 10 min for each wash with rotation at 4°C.
6. Elute the protein with 5 to 6 bed volumes of Py-Elution Buffer/50 μg/ml Peptide. Rotate for 2 hr at 4°C.
7. Elute a second time with Py-Elution Buffer/100 μg/ml Peptide (see Hint #4).
8. Check for the presence of the protein of interest in the fractions on a Polyacrylamide gel (see Protocol on Running Polyacrylamide Gels). If the protein recovery was poor, try using an elution solution containing up to 500 μg/ml peptide to elute the protein (see Hints #5 and #6).
9. The column can be recycled by washing it with 5 volumes of Carbonate Buffer (see Hint #7).
|
| Py-Elution Buffer |
| 5% (v/v) Glycerol
0.1 mM DTT
100 to 300 mM NaCl
0.1% (v/v) Igepal CA-630
0.2 mM PMSF
25 mM HEPES-KOH
|
 |
| Py-Wash Buffer |
| 0.1 mM DTT
100 mM NaCl
0.1 mM PMSF
0.5% (v/v) Igepal CA-630
20 mM HEPES-KOH
2.5% (v/v) Glycerol
0.1 mM EDTA
|
|
| High pH Elution Buffer |
| 0.05% (v/v) Igepal CA-630
Check that the pH is approximately 11.0
200 mM NaCl
10% (v/v) Glycerol
20 mM Triethylamide
|
|
| Carbonate Buffer |
| 100 mM Na2CO3
0.5% (v/v) Igepal CA-630
|
|
| Py-Elution Buffer/100 μg/ml Peptide |
| 8 ul of 500X Peptide Stock
in 2 ml Elution Buffer
|
|
| Py-Elution Buffer/50 μg/ml Peptide |
| 4 ul of 500X Peptide Stock
in 2 ml Elution Buffer
|
|
| Peptide Stock (500X) |
| 25 mg/ml Peptide (EYMPME)
Keep a working stock at 4°C to avoid freeze and thaw cycles that cause the stock to precipitate.
|
|
|
| |
Potassium Hydroxide
Triethylamine
PMSF
IGEPAL CA-630
Sodium Chloride
Sodium Carbonate
Sodium Azide
Glycerol
Dimethylpimelimidate
HEPES
Peptide (EYMPME)
EDTA
Anti-Py Antibody-Protein-G Sepharose
DTT
N-Octylglucoside
|
| |
1. For longer storage periods, add Sodium Azide to 0.02% (w/v). CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Only small amounts of beads are required. In theory, one ml of resin should be able to bind about 6 to 12 mg of an 87 kDa protein.
3. For larger scale purifications, prepare the column and load the supernatant slowly or several times onto one column.
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 - 1 M NaCl). Follow with a lower salt dialysis buffer if necessary. You can also run additional columns.
5. Higher peptide concentrations will not give any improvement for this particular peptide. Adding higher salt, or 0.1% (v/v) N-Octylglucoside, and/or higher Igepal CA-630 concentration to the Elution Buffer may improve the elution.
6. High pH elution can be used instead of Step #6. For batch-wise elution, add 4 bead volumes of High pH Elution Buffer and incubate for 10 sec while mixing well. Centrifuge briefly in a microcentrifuge at full speed. Transfer the supernatant to a new tube containing 0.05 volume of 1 M HEPES-KOH, pH 7.6 to neutralize. Repeat the elution 5 times. For columns, elute with at least 10 column volumes of High pH Elution Buffer and catch fractions in tubes containing 0.05 volumes of 1 M HEPES-KOH, pH 7.6 to neutralize the fractions. A pH lower than 11 does not work; only try higher pH if elution is not satisfactory. Carbonate buffer at pH 11 does not work as well as the Triethylamine buffer
7. 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.
|
