Altman:Protocols/Protein Purification/SF9 purification

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(New page: Prepare the following beforehand: Lysis Buffer 200 mM NaCl 4 mM MgCl2 20 mM Imidazole, pH 7.5 0.5 mM EDTA 1 mM EGTA 0.5% Igepal 7% sucrose 1 mM PMSF (0.25 M stock) 10 μg/mL Aprot...)
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Prepare the following beforehand:
Prepare the following beforehand:
-
Lysis Buffer
+
* LYSIS BUFFER
-
 
+
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% Igepal
-
7% sucrose
+
:7% 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
 +
 
 +
{{Template:Altman}}
 +
 
 +
<div style="padding: 10px; width: 700px; border: 5px solid #B22222;">
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 +
==Making Glucose Oxidase Catalase==
 +
 
 +
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">
 +
 
 +
<tr>
 +
<th><tt> </tt></th>
 +
<th><tt>1 mL 2xAB </tt></th>
 +
</tr>
 +
 
 +
<tr>
 +
<th><tt> 10x AB stock </tt></th>
 +
<th><tt> 200 uL </tt></th>
 +
</tr>
 +
 
 +
<tr>
 +
<th><tt> 100x DTT stock </tt></th>
 +
<th><tt> 20 uL </tt></th>
 +
</tr>
 +
 
 +
<tr>
 +
<th><tt> ddwater </tt></th>
 +
<th><tt> 780 uL </tt></th>
 +
</tr>
 +
 
 +
</table>
 +
 
 +
2. Make 43 mg/mL glucose oxidase (GO) in 2xAB.
 +
: Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.
 +
3. Make 7.2 mg/mL catalase.
 +
: Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.
-
1 mL Assay Buffer
+
4. Add glycerol to the GO and catalase stocks 1:1 volume/volume.
 +
: Add 400 μL of glycerol to each, being careful to not introduce bubbles, especially in the GO.
 +
: This produces 100x stocks of GO and catalase.
-
(AB)
+
5. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC.
-
1 mL Assay Buffer + BSA
+
-
(ABSA)
+
6. Aliquot into 50 μL stocks, which are stored at 20°C
-
10x AB stock 100 uL 100 uL
+
-
100x DTT stock 10 uL 10 uL
+
-
10x BSA stock -- 100 uL
+
-
ddwater 890 uL 790 uL
+

Revision as of 13:01, 19 February 2013


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% Igepal
7% sucrose
1 mM PMSF (0.25 M stock)
10 μg/mL Aprotinin
10 μg/mL Leupeptin
5 mM DTT
2 mM ATP


Department of Physics, Willamette University

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Making Glucose Oxidase Catalase

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)

1 mL 2xAB
10x AB stock 200 uL
100x DTT stock 20 uL
ddwater 780 uL

2. Make 43 mg/mL glucose oxidase (GO) in 2xAB.

Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.

3. Make 7.2 mg/mL catalase.

Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.

4. Add glycerol to the GO and catalase stocks 1:1 volume/volume.

Add 400 μL of glycerol to each, being careful to not introduce bubbles, especially in the GO.
This produces 100x stocks of GO and catalase.

5. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC.

6. Aliquot into 50 μL stocks, which are stored at 20°C
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