Overview

  • Product name

    PDE Activity Assay Kit (Colorimetric)
  • Detection method

    Colorimetric
  • Sample type

    Tissue, Inhibitor compounds
  • Assay type

    Enzyme activity
  • Assay time

    0h 30m
  • Product overview

    PDE Activity Assay Kit (Colorimetric) ab139460 combines a special dual enzyme system with Green Assay Reagent for phosphate detection to create a unique, non-radioactive, colorimetric assay to detect phosphodiesterase (PDE) activity. This HTS-friendly, mix and read system may be used to screen inhibitors and modulators of cyclic nucleotide phosphodiesterase activity. 96-well microplate format permits rapid assays of large numbers of samples. The basis for the assay is the cleavage of cAMP or cGMP by a cyclic nucleotide phosphodiesterase.


    The kit includes a Type I PDE as positive control and a non-specific PDE inhibitor, 2-isobutyl-1-methylzanthine (IBMX) as test control for inhibitor screening.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests
    3',5'-cAMP Substrate 1 x 2ml
    3',5'-cGMP Substrate 1 x 2ml
    5' AMP Standard 1 x 1ml
    5'-GMP Standard 1 x 1ml
    5'-Nucleotidase (from Crotalus atrox venom) 1 x 1ml
    96-well Clear Microplate (1/2 Volume) 1 unit
    Desalting Column 1 unit
    Desalting Resin 1 x 1g
    Green Assay Reagent 1 x 20ml
    PDE Assay Buffer 1 x 40ml
    PDE Enzyme (from bovine brain) 5 vials
    PDE Inhibitor 1 x 200µl
  • Research areas

  • Relevance

    3'5'-cyclic nucleotide phosphodiesterases are a family of phosphodiesterases. Generally, these enzymes hydrolyze a nucleoside 3’,5’-cyclic phosphate to a nucleoside 5’-phosphate. Some examples of nucleoside 3’,5’-cyclic phosphate include: 3',5'-cyclic AMP, 3',5'-cyclic dAMP, 3',5'-cyclic IMP, 3',5'-cyclic GMP, 3',5'-cyclic CMP

Images

  • Duplicate wells of 5’-AMP dilutions were prepared. Phosphate was released from 5’-AMP by incubation with 5’-nucleotidase (50 kU/well, 30°C, 30 min.) and the reaction terminated by addition of Green Assay Reagent (100 µL/well). After 30 min., the phosphate-dependent color reaction was measured by reading OD620nm in a microplate-reading spectrophotometer.

  • PDE enzyme (20 mU/well) was incubated with cAMP (200 μM) and 5’-nucleotidase (50 kU/well) with or without the inhibitor IBMX (40 μM) at 30°C for the indicated times. Reactions were terminated by addition of 100 µL of Green Assay Reagent and OD620nm read 30 min. later. A cAMP standard curve may be used to convert OD620nm data to nmol of 5’-AMP.

Protocols

References

This product has been referenced in:

  • Araiz C  et al. Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110a inactivation in adipose tissue. Nat Commun 10:1546 (2019). Read more (PubMed: 30948720) »
  • Mukohda M  et al. RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity. J Clin Invest 130:2318-2332 (2019). Read more (PubMed: 30896450) »
See all 13 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer



5'-Nucleotidase (from Crotalus atrox venom) is available to purchase separately and can be used as a positive control. This has been added to the catalog for you:

ab207796
https://www.abcam.com/index.html?datasheet=207796 (or use the following: https://www.abcam.com/index.html?datasheet=207796).

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Answer

is indeed possible to use the desalting column and resin for multiple samples. After each sample, the column should be rinsed with several volumes of phosphate free water, and then re-equilibrated with assay buffer (return to step 4 of the desalting protocol on page 6) before moving on to the next sample.

Exactly how many samples can be processed will vary depending upon the samples in question and the amount of free phosphate present. The column will eventually start to lose efficiency (and samples may have to be run through twice in order to remove all the free phosphate). Again, the rate at which efficiency is lost can vary depending on the samples used. When this happens the exhausted resin should be discarded. Normally it is somewhere in the range of 5-7 samples. (Note : enough resin is provided to fill the column twice, so a total of 10-14 samples on average can be processed with the materials provided in the kit).

you can substitute any size-exclusion spin columns with a molecular weight cut-off of 6000, which may be a more convenient alternative for multiple samples. We recommend Centrispin 10 columns (CS-100 or CS-101) from Princeton Separations (http://www.prinsep.com/ ) or Zebaspin columns from Pierce (http://www.piercenet.com/products/browse.cfm?fldID=67687BF8-62AB-4FBD-A404-BEC6E0635687).

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Answer

The ab139460 kit can be used to assess the activity of PDE in cell or tissue lysates, as well as with purified enzyme. However we do not have any specific buffer formulations to offer for we have not optimized one for this assay. You can use any lysis buffer provided it does not effect the activity of enzyme - avoid urea, tris etc. in buffer. While it will also be important to not include reagents that will inhibit the reaction, it will be most important to make sure you do not introduce additional free phosphates for the Biomol green will falsely pick these up as additional relevant signal.

Cell or tissue lysates will have to be desalted to remove endogenous free phosphate prior to assay. The kit includes one set of desalting column and resin which should be good for a total of 12-18 samples (there is enough resin provided to fill the column twice, and each batch of resin is good for 6-9 samples depending upon how much free phosphate is present). If the customer has more than 18 samples they will need to either desalt through a different method, or purchase additional sets of desalting column and resin. Instructions for desalting lysates are included in the protocol provided.

Please note: Lysates may need to be diluted before assay as the levels of PDE activity vary widely depending upon the cell or tissue type. The customer will have to do some initial experimentation to determine if their particular samples need to be diluted (or possibly concentrated) in order to produce results within the range of the standard curve.

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