Altman:Protocols/Protein Purification/SF9 purification

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Line 9: Line 9:
* LYSIS BUFFER
* LYSIS BUFFER
-
Final concentrations:  
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:Final concentrations:  
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:200 mM NaCl
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::200 mM NaCl
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:4 mM MgCl2
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::4 mM MgCl2
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:20 mM Imidazole, pH 7.5
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::20 mM Imidazole, pH 7.5
-
:0.5 mM EDTA
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::0.5 mM EDTA
-
:1 mM EGTA
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::1 mM EGTA
-
:0.5% Igepal
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::0.5% (v/v) Igepal  
-
:7% sucrose
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::7% (w/v) sucrose  
-
:1 mM PMSF (0.25 M stock)
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::1 mM PMSF (0.25 M stock)
-
:10 μg/mL Aprotinin  
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::10 μg/mL Aprotinin  
-
:10 μg/mL Leupeptin  
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::10 μg/mL Leupeptin  
-
:5 mM DTT   
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::5 mM DTT   
-
:2 mM ATP
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::2 mM ATP
-
 
+
-
 
+
-
FINAL CONCENTRATION of 50x GOC stock: 10.8 mg/mL glucose oxidase, 1.8 mg/mL catalase
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in 1x AB-DTT and 50% Glycerol
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-
 
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* Glucose Oxidase, Aspergillus niger (Calbiochem, 346385)
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* Catalase, from Bovine Liver (Sigma, 19.9 mg/mL)
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* Glycerol, Anhydrous
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-
 
+
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1. Make 2x Assay Buffer (2xAB)
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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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<tr>
<tr>
<th><tt> </tt></th>
<th><tt> </tt></th>
-
<th><tt>1 mL 2xAB </tt></th>
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<th><tt>5 mL total </tt></th>
</tr>
</tr>
<tr>
<tr>
-
<th><tt> 10x AB stock </tt></th>
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<th><tt> 2x lysis buffer </tt></th>
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<th><tt> 200 uL </tt></th>
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<th><tt> 2.5 mL </tt></th>
</tr>
</tr>
<tr>
<tr>
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<th><tt> 100x DTT stock </tt></th>
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<th><tt> 1 mg/mL aprotinin stcok </tt></th>
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<th><tt> 20 uL </tt></th>
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<th><tt> 50 uL </tt></th>
</tr>
</tr>
<tr>
<tr>
-
<th><tt> ddwater </tt></th>
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<th><tt> 1 mg/mL leupeptin stock </tt></th>
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<th><tt> 780 uL </tt></th>
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<th><tt> 50 uL </tt></th>
</tr>
</tr>
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</table>
 
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2. Make 43 mg/mL glucose oxidase (GO) in 2xAB.
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<tr>
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: Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.
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<th><tt> 1 M DTT </tt></th>
 +
<th><tt> 25 uL </tt></th>
 +
</tr>
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3. Make 7.2 mg/mL catalase.
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<tr>
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: Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.
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<th><tt> 100 mM ATP  </tt></th>
 +
<th><tt> 100 uL </tt></th>
 +
</tr>
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4. Add glycerol to the GO and catalase stocks 1:1 volume/volume.
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<tr>
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: Add 400 μL of glycerol to each, being careful to not introduce bubbles, especially in the GO.
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<th><tt> 0.25 M PMSF </tt></th>
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: This produces 100x stocks of GO and catalase.
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<th><tt> 20 uL </tt></th>
 +
</tr>
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5. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC.
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<tr>
 +
<th><tt> diwater </tt></th>
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<th><tt> 2.255 mL </tt></th>
 +
</tr>
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6. Aliquot into 50 μL stocks, which are stored at 20°C
+
</table>

Revision as of 13:06, 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
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
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