Altman:Protocols/Protein Purification/SF9 purification: Difference between revisions
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* LYSIS BUFFER | * LYSIS BUFFER | ||
Final concentrations: | ** 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. | ||
:200 mM NaCl | :Final concentrations: | ||
:4 mM MgCl2 | ::200 mM NaCl | ||
:20 mM Imidazole, pH 7.5 | ::4 mM MgCl2 | ||
:0.5 mM EDTA | ::20 mM Imidazole, pH 7.5 | ||
:1 mM EGTA | ::0.5 mM EDTA | ||
:0.5% Igepal | ::1 mM EGTA | ||
:7% | ::0.5% (v/v) Igepal | ||
:1 mM PMSF (0.25 M stock) | ::7% (w/v) Sucrose | ||
:10 μg/mL Aprotinin | ::1 mM PMSF (0.25 M stock) | ||
:10 μg/mL Leupeptin | ::10 μg/mL Aprotinin | ||
:5 mM DTT | ::10 μg/mL Leupeptin | ||
:2 mM ATP | ::5 mM DTT | ||
::2 mM ATP | |||
<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | |||
<tr> | |||
<th><tt> </tt></th> | |||
<th><tt>5 mL total </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 2x lysis buffer </tt></th> | |||
<th><tt> 2.5 mL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 1 mg/mL aprotinin stock </tt></th> | |||
<th><tt> 50 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 1 mg/mL leupeptin stock </tt></th> | |||
<th><tt> 50 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 1 M DTT </tt></th> | |||
<th><tt> 25 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 100 mM ATP </tt></th> | |||
<th><tt> 100 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> 0.25 M PMSF </tt></th> | |||
<th><tt> 20 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> diwater </tt></th> | |||
<th><tt> 2.255 mL </tt></th> | |||
</tr> | |||
</table> | |||
1 | * 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 | |||
<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | <table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | ||
Line 37: | Line 83: | ||
<tr> | <tr> | ||
<th><tt> </tt></th> | <th><tt> </tt></th> | ||
<th><tt> | <th><tt>20 mL total </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <th><tt> 5 M NaCl </tt></th> | ||
<th><tt> | <th><tt> 1.6 mL </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <th><tt> 1 M MgCl2 </tt></th> | ||
<th><tt> | <th><tt> 160 uL </tt></th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th><tt> | <th><tt> I M Imidazole, pH 7.5 </tt></th> | ||
<th><tt> | <th><tt> 800 uL </tt></th> | ||
</tr> | </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> |
Latest revision as of 10:46, 19 February 2013
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 |