Phosphodiesterase II - Assay

Source:
Bovine Spleen
CAS:
9068-54-6
EC:
3.1.16.1

Phosphodiesterase II Assay

Method
A modification of the assay as described by Hilmoe (1961). One unit is defined as a change in absorbance of 0.2 at 37°C and pH 6.5 under the specified conditions.
Reagents
  • 5.9 mM Uranyl acetate in 2.5% perchloric acid. Prepare by dissolving 250 mg uranyl acetate and 3.55 ml 70% perchloric acid in 80 ml reagent grade water. Dilute to a final volume of 100 ml with reagent grade water.
  • 0.25 M Sodium succinate⋅HCl, pH 6.5
  • Worthington RNA Core (Code:RNAC). Dissolve at a concentration of 17 mg/ml in reagent grade water. Adjust pH to 7.0.
Enzyme

Dissolve contents of vial with one ml reagent grade water. Prior to use, dilute to 2-8 units/ml with reagent grade water.

Procedure

Prepare a 37°C water bath.

Pipette into tubes as follows:

  Test Blank
Sodium succinate buffer 0.4 ml 0.4 ml
RNA core 0.5 ml 0.5 ml
Reagent grade water 1.0 ml 1.1 ml
Incubate tubes in 37°C water bath for 5 minutes to achieve temperature equilibration. At timed intervals add:
Enzyme dilution 0.1 ml ------

Incubate at 37°C for 30 minutes. Stop the reaction at timed intervals by adding 2.0 ml uranyl acetate/perchloric acid solution. Remove to an ice bath and chill for 10 minutes. Centrifuge at high speed in a clinical centrifuge for 15 minutes. Transfer 0.2 ml aliquot of each supernatant to a clean dry test tube and add 7.8 ml reagent grade water. Mix well and read A260 vs water.

Calculation

Phosphodiesterase II Products

Description
Activity
Code
Cat. #
Size
Price
Description
Phosphodiesterase II
Source:
Bovine Spleen
Prepared from the 1mM sodium pyrophosphate, pH 6.9, alumina gel eluate of Hilmoe: Biochem. Prep VIII (Meister, A., ed.), John Wiley & Sons, NY, NY, p. 105 (1961).
Store at -20°C.
Ice Pack required
Activity
≥1.2 u/mg dw
Code
SPH
LS003603
10 un
$118.00
LS003602
25 un
$265.00
LS003600
Bulk
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The minimum amount for bulk packaging/pricing for this product is 500 x 1 un. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.