Altman:Protocols/Preparing Reagents/Making GOC: Difference between revisions

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* Glycerol, Anhydrous
* Glycerol, Anhydrous


1. Make 2xAB-DTT (10x AB stock and the 100x DTT stock in half the usual AB volume)  
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">
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<tr>
<tr>
<th><tt> </tt></th>
<th><tt> </tt></th>
<th><tt>1 mL 2xAB-DTT </tt></th>
<th><tt>1 mL 2xAB </tt></th>
</tr>
</tr>


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<th><tt> 100x DTT stock </tt></th>
<th><tt> 100x DTT stock </tt></th>
<th><tt> 20 uL </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>


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</table>
</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.


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.


1. Make 43 mg/mL glucose oxidase (GO) in 2xAB.
6. Aliquot into 50 μL stocks, which are stored at 20°C
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.

Revision as of 22:46, 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 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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