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

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==Small-scale purification of FLAG-tagged proteins from SF9 cells==




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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% sucrose
::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>


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<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>


==Making Glucose Oxidase Catalase==
<tr>
<th><tt> 0.25 M PMSF </tt></th>
<th><tt> 20 uL </tt></th>
</tr>


FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase
<tr>
in 1x AB-DTT and 50% Glycerol
<th><tt> diwater </tt></th>
<th><tt> 2.255 mL </tt></th>
</tr>


* Glucose Oxidase, Aspergillus niger (Calbiochem, 346385)
</table>
* Catalase, from Bovine Liver (Sigma, 19.9 mg/mL)
* Glycerol, Anhydrous


1. Make 2x Assay Buffer (2xAB)
* 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


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<tr>
<tr>
<th><tt> </tt></th>
<th><tt> </tt></th>
<th><tt>1 mL 2xAB </tt></th>
<th><tt>20 mL total </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 10x AB stock </tt></th>
<th><tt> 5 M NaCl </tt></th>
<th><tt> 200 uL </tt></th>
<th><tt> 1.6 mL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> 100x DTT stock </tt></th>
<th><tt> 1 M MgCl2 </tt></th>
<th><tt> 20 uL </tt></th>
<th><tt> 160 uL </tt></th>
</tr>
</tr>


<tr>
<tr>
<th><tt> ddwater </tt></th>
<th><tt> I M Imidazole, pH 7.5 </tt></th>
<th><tt> 780 uL </tt></th>
<th><tt> 800 uL </tt></th>
</tr>
</tr>


</table>


2. Make 43 mg/mL glucose oxidase (GO) in 2xAB.
<tr>
: Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.
<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>


3. Make 7.2 mg/mL catalase.
<tr>
: Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.
<th><tt> Igepal </tt></th>
<th><tt> 200 uL </tt></th>
</tr>


4. Add glycerol to the GO and catalase stocks 1:1 volume/volume.
<tr>
: Add 400 μL of glycerol to each, being careful to not introduce bubbles, especially in the GO.
<th><tt> Sucrose </tt></th>
: This produces 100x stocks of GO and catalase.
<th><tt> 2.8 g </tt></th>
</tr>


5. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC.
<tr>
<th><tt> diwater </tt></th>
<th><tt> raise to 20 mL </tt></th>
</tr>


6. Aliquot into 50 μL stocks, which are stored at 20°C
</table>

Latest revision as of 10:46, 19 February 2013


Department of Physics, Willamette University

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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
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