Altman:Protocols/Protein Purification/SF9 purification
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<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | <table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | ||
| + | |||
| + | * 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 | ||
| + | |||
<tr> | <tr> | ||
Revision as of 13:07, 19 February 2013
Department of Physics, Willamette University
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 |
