Difference between revisions of "Altman:Protocols/Protein Purification/SF9 purification"

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* LYSIS BUFFER
 
* 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:  
 
:Final concentrations:  
 
::200 mM NaCl
 
::200 mM NaCl
Line 16: Line 17:
 
::1 mM EGTA
 
::1 mM EGTA
 
::0.5% (v/v) Igepal  
 
::0.5% (v/v) Igepal  
::7% (w/v) sucrose
+
::7% (w/v) Sucrose
 
::1 mM PMSF (0.25 M stock)
 
::1 mM PMSF (0.25 M stock)
 
::10 μg/mL Aprotinin  
 
::10 μg/mL Aprotinin  
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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>
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<tr>
 
<tr>
<th><tt> 1 mg/mL aprotinin stcok </tt></th>
+
<th><tt> 1 mg/mL aprotinin stock </tt></th>
 
<th><tt> 50 uL </tt></th>
 
<th><tt> 50 uL </tt></th>
 
</tr>
 
</tr>
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<th><tt> diwater </tt></th>
 
<th><tt> diwater </tt></th>
 
<th><tt> 2.255 mL </tt></th>
 
<th><tt> 2.255 mL </tt></th>
 +
</tr>
 +
 +
</table>
 +
 +
* 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">
 +
 +
<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>
 
</tr>
  
 
</table>
 
</table>

Latest revision as of 09:46, 19 February 2013

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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