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"> | ||
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::1% (v/v) Igepal | ::1% (v/v) Igepal | ||
::14% (w/v) sucrose | ::14% (w/v) sucrose | ||
| + | |||
| + | |||
| + | <table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> </tt></th> | ||
| + | <th><tt>20 mL total </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> 5 M NaCl </tt></th> | ||
| + | <th><tt> 1.6 mL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> 1 M MgCl2 </tt></th> | ||
| + | <th><tt> 160 uL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> I M Imidazole, pH 7.5 </tt></th> | ||
| + | <th><tt> 800 uL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | |||
| + | <tr> | ||
| + | <th><tt> 0.5 M EDTA </tt></th> | ||
| + | <th><tt> 40 uL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> 0.5 M EGTA </tt></th> | ||
| + | <th><tt> 80 uL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> Igepal </tt></th> | ||
| + | <th><tt> 200 uL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> Sucrose </tt></th> | ||
| + | <th><tt> 2.8 g </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <th><tt> diwater </tt></th> | ||
| + | <th><tt> raise to 20 mL </tt></th> | ||
| + | </tr> | ||
| + | |||
| + | </table> | ||
Revision as of 13:43, 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 |
- 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 |
