Altman:Protocols/Protein Purification/SF9 purification
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Department of Physics, Willamette University
Small-scale purification of FLAG-tagged proteins from SF9 cells
Prepare the following beforehand:
- LYSIS BUFFER
- NOTE: if you are freezing down cells before the purification, leave out the Igepal and Sucrose. This will require you to make 2x lysis buffer without these two ingredients.
- Final concentrations:
- 200 mM NaCl
- 4 mM MgCl2
- 20 mM Imidazole, pH 7.5
- 0.5 mM EDTA
- 1 mM EGTA
- 0.5% (v/v) Igepal
- 7% (w/v) Sucrose
- 1 mM PMSF (0.25 M stock)
- 10 μg/mL Aprotinin
- 10 μg/mL Leupeptin
- 5 mM DTT
- 2 mM ATP
| 5 mL total | |
|---|---|
| 2x lysis buffer | 2.5 mL |
| 1 mg/mL aprotinin stock | 50 uL |
| 1 mg/mL leupeptin stock | 50 uL |
| 1 M DTT | 25 uL |
| 100 mM ATP | 100 uL |
| 0.25 M PMSF | 20 uL |
| diwater | 2.255 mL |
- 2x LYSIS BUFFER
- Final Concentrations
- 400 mM NaCl
- 8 mM MgCl2
- 40 mM Imidazole, pH 7.5
- 1 mM EDTA
- 2 mM EGTA
- 1% (v/v) Igepal
- 14% (w/v) sucrose
| 20 mL total | |
|---|---|
| 5 M NaCl | 1.6 mL |
| 1 M MgCl2 | 160 uL |
| I M Imidazole, pH 7.5 | 800 uL |
| 0.5 M EDTA | 40 uL |
| 0.5 M EGTA | 80 uL |
| Igepal | 200 uL |
| Sucrose | 2.8 g |
| diwater | raise to 20 mL |
