The enzyme assay is performed with Peroxidase Assay Reagent (1.0 mM ABTS and 1.2

Question

The enzyme assay is performed with Peroxidase Assay Reagent (1.0 mM ABTS and 1.2 mM H2O2 in sodium acetate buffer). How would you prepare 25 mL of enzyme assay reagent from the listed stock solutions?
Supplies/Stock Solutions:
1. Sodium acetate buffer, 100.0 mM, pH 6.0
2. NaCl
3. Mannose (MW: 180.2)
4. Horseradish root, turnip or horseradish peroxidase (in 1% milk in sodium acetate, diluted 1/20)
5. 50 mM stock ABTS (made in water) – keep the stock ABTS on ice
6. 30% H2O2 stock (8.8 M) – keep the stock H2O2 on ice (caution: 30% H2O2 can cause chemical burns)
7. Peroxidase Assay Reagent – 1.0 mM ABTS and 1.2 mM hydrogen peroxide in sodium acetate buffer – the assay reagent may be kept at room temperature
8. Bio-Rad Bradford dye reagent
9. Standard protein, bovine serum albumin (BSA)
10. Your samples isolated from roots of horseradish or turnip

Explanation / Answer

We will have to prepare 1.0 mM ABTS from stock of 50 mM ABTS.

Therefore applying V1S1=V2S2 formula,

50mM x V1 =1.0 mM x 1ml             So, V1 =1.0 x 1 /50 = 0.02 ml = 20 microlitre     (since 1ml= 1000 microlitre)

So 20 microlitre of 50mM ABTS is taken and the volume is made upto 1 ml with the buffer so that the final prepared solution is 1 ml 1 mM ABTS solution.

Next, We will have to prepare 1.2 mM H2O2 from stock of 8.8 M of 30% H2O2 solution.

Therefore applying V1S1=V2S2 formula,

8.8 M x V1 =1.2 mM x 1ml      =>> 8800 mM X V1 = 1.2 mM x 1ml       (since 1M= 1000 mM)

V1 =  1.2 mM x 1ml / 8800 mM ==>>V1 = 1.36 x 10-4 ml = 0.136 microlitre.                                          

(since 1ml= 1000 microlitre)

So, 0.136 microlitre of 8.8 M H2O2 solution is taken and the volume is made upto 1 ml with the buffer so that the final prepared solution is 1 ml 1.2 mM H2O2 solution.

Total volume of assay reagent = 25 ml

So sodium acetate buffer added = 25-(1+1)=25 - 2 = 23 ml

(I have done the calculation that to prepare 25 ml of assay reagent I need to add 1 ml of 1.0 mM ABTS and 1 ml of 1.2 mM H2O2 since the volume of this two that is ABTS and H2O2 are not given in the final assay reagent,so I have done the calculation considering that).(It is given ---7. Peroxidase Assay Reagent – 1.0 mM ABTS and 1.2 mM hydrogen peroxide in sodium acetate buffer , so only concentrations are provided but not volume so I have taken each volume to be 1 ml)

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