Altman:Protocols/Protein Purification/SF9 purification: Difference between revisions
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* 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 |
Revision as of 10:08, 19 February 2013
Small-scale purification of FLAG-tagged proteins from SF9 cells
Prepare the following beforehand:
- LYSIS BUFFER
- 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 stcok | 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