For our international customers, please be advised that orders cannot be placed through our website by customers in countries with International Distributor representation.
Phosphodiesterase I - Assay
Source:
Crotalus adamanteus Venom
CAS:
9025-82-5
EC:
3.1.4.1
Phosphodiesterase I Assay
Method
The assay is essentially that of Razell and Khorana (1959) where the reaction velocity is determined by an increase in absorbance at 400 nm resulting from the hydrolysis of p-nitrophenyl thymidine-5'-phosphate. One unit hydrolyzes one micromole of p-nitrophenyl thymidine-5'-phosphate per minute at pH 8.9 and 25°C under the specified conditions.
Reagents
- 0.11 M Tris⋅HCl buffer, pH 8.9, with 0.11 M NaCl and 15 mM MgCl2 (Tris⋅Salts buffer)
- 5 mM p-nitrophenyl thymidine-5'-phosphate. Note: The purity of commercial preparations varies somewhat and should be considered in preparing this reagent.
Enzyme
Dissolve at one mg/ml in Tris*Salts buffer to obtain a rate of 0.02-0.04 ΔA/minute.
Procedure
Set spectrophotometer at 400 nm and 25°C. Pipette into microcuvettes as follows:
Tris⋅Salts buffer 0.9 ml 5 mM p-nitrophenyl thymidine-5'-phosphate 0.1 ml
Incubate cuvettes in spectrophotometer for 3-5 minutes to reach temperature equilibrium and establish blank rate, if any. Add 10 microliters of diluted enzyme and record increase in A400 for 3-5 minutes. The reaction remains linear until A400 reaches about 1.2. Calculate ΔA400/minute from initial linear portion of absorbance curve.
Calculation
Phosphodiesterase I Products
Description
Activity
Code
Cat. #
Size
Price
Description
Phosphodiesterase I
Source:
Crotalus adamanteus Venom
Purified by the method of Williams, Sung and Laskowski, JBC, 236, 1130 (1961). Further treated to inactivate contaminating 5'-nucleotidase activity according to Sulkowski and Laskowski: BBA, 240, 443 (1961). Lyophilized in vials.
Store at -20°C.
Ice Pack required
Code
VPH
Product details
LS003926
100 un
$104.00
LS003928
Bulk
---