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

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* LYSIS BUFFER
 
* LYSIS BUFFER
Final concentrations:  
+
:Final concentrations:  
:200 mM NaCl
+
::200 mM NaCl
:4 mM MgCl2
+
::4 mM MgCl2
:20 mM Imidazole, pH 7.5
+
::20 mM Imidazole, pH 7.5
:0.5 mM EDTA
+
::0.5 mM EDTA
:1 mM EGTA
+
::1 mM EGTA
:0.5% Igepal
+
::0.5% (v/v) Igepal  
:7% 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  
:10 μg/mL Leupeptin  
+
::10 μg/mL Leupeptin  
:5 mM DTT   
+
::5 mM DTT   
:2 mM ATP
+
::2 mM ATP
 
 
 
 
FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase
 
in 1x AB-DTT and 50% Glycerol
 
 
 
* Glucose Oxidase, Aspergillus niger (Calbiochem, 346385)
 
* Catalase, from Bovine Liver (Sigma, 19.9 mg/mL)
 
* Glycerol, Anhydrous
 
 
 
1. Make 2x Assay Buffer (2xAB)
 
  
 
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
 
<table id="Table 1" border="1" cellpadding="3" cellspacing="0">
Line 37: Line 27:
 
<tr>
 
<tr>
 
<th><tt> </tt></th>
 
<th><tt> </tt></th>
<th><tt>1 mL 2xAB </tt></th>
+
<th><tt>5 mL total </tt></th>
 
</tr>
 
</tr>
  
 
<tr>
 
<tr>
<th><tt> 10x AB stock </tt></th>
+
<th><tt> 2x lysis buffer </tt></th>
<th><tt> 200 uL </tt></th>
+
<th><tt> 2.5 mL </tt></th>
 
</tr>
 
</tr>
  
 
<tr>
 
<tr>
<th><tt> 100x DTT stock </tt></th>
+
<th><tt> 1 mg/mL aprotinin stcok </tt></th>
<th><tt> 20 uL </tt></th>
+
<th><tt> 50 uL </tt></th>
 
</tr>
 
</tr>
  
 
<tr>
 
<tr>
<th><tt> ddwater </tt></th>
+
<th><tt> 1 mg/mL leupeptin stock </tt></th>
<th><tt> 780 uL </tt></th>
+
<th><tt> 50 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> 1 M DTT </tt></th>
 +
<th><tt> 25 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> 100 mM ATP  </tt></th>
 +
<th><tt> 100 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> 0.25 M PMSF </tt></th>
: This produces 100x stocks of GO and catalase.
+
<th><tt> 20 uL </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> 2.255 mL </tt></th>
 +
</tr>
  
6. Aliquot into 50 μL stocks, which are stored at 20°C
+
</table>

Revision as of 10:06, 19 February 2013

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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