Altman:Protocols/Preparing Reagents/Making GOC: Difference between revisions
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in 1x AB-DTT and 50% Glycerol | in 1x AB-DTT and 50% Glycerol | ||
* Glucose Oxidase, Aspergillus niger | * Glucose Oxidase, Aspergillus niger (Calbiochem, 346385) | ||
* Catalase, from Bovine Liver (Sigma, 19.9 mg/mL) | |||
* Catalase, from Bovine Liver | |||
* Glycerol, Anhydrous | * Glycerol, Anhydrous | ||
1. Make 2xAB-DTT (10x AB stock and the 100x DTT stock in half the usual AB volume) | |||
<table id="Table 1" border="1" cellpadding="3" cellspacing="0"> | |||
1 mL | |||
10x AB stock | <tr> | ||
100x DTT stock | <th><tt> </tt></th> | ||
<th><tt>1 mL 2xAB-DTT </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> 10x BSA stock </tt></th> | |||
<th><tt> -- </tt></th> | |||
<th><tt> 100 uL </tt></th> | |||
</tr> | |||
<tr> | |||
<th><tt> ddwater </tt></th> | |||
<th><tt> 780 uL </tt></th> | |||
</tr> | |||
</table> | |||
1. Make 43 mg/mL glucose oxidase (GO) in 2xAB. | 1. Make 43 mg/mL glucose oxidase (GO) in 2xAB. | ||
Revision as of 22:43, 14 November 2012
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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 2xAB-DTT (10x AB stock and the 100x DTT stock in half the usual AB volume)
| 1 mL 2xAB-DTT | ||
|---|---|---|
| 10x AB stock | 200 uL | |
| 100x DTT stock | 20 uL | |
| 10x BSA stock | -- | 100 uL |
| ddwater | 780 uL |
1. Make 43 mg/mL glucose oxidase (GO) in 2xAB. Combine 17 mg of GO with 400 μL 2xAB to make ~400 μL of GO.
2. Make 7.2 mg/mL catalase. Combine 145 μL of catalase with 255 μL of 2xAB to make 400 μL of catalase.
3. Add glycerol to sample 1:1 volume/volume. Added 400 μL of glycerol to each. NOTE: be careful to not introduce bubbles, especially in the GO. This produces 100x stocks of GO and catalase.
4. Combine the GO stock and catalase stock 1:1 volume/volume to yield a 50x stock of GOC. Combined 700 μL of each Aliquoted into 50 μL stocks which are stored at 20°C (in Ron’s freezer. Do not freeze. Made 27 aliquots.
