PDEase Kit Protocol

 

Preparation of incubation buffers and assay set up

 

1. Incubation buffer must be prepared by mixing the stock reagents immediately before use in assay
2. Place duplicate rows of 1.5 ml plastic conical tubes with snap lock caps on ice.  Alternatively, a small hole can be punctured using 16-18 gauge needle for venting the steam that will generate in subsequent steps.
3. Pipette 25 µl of incubation buffer prepared by mixing incubation buffer Reagents A, B and C (3:1:1 v/v with A:B and C) in each conical tube placed on ice at 4⁰C.  Prepare this buffer immediately prior to use.
4. Dilute the PDE enzyme in incubation buffer (prepared in step 2) to get desired PDE activity in 25 µl of incubation buffer. Generally 25-50 µg protein from cell extracts.
5. Add 25 µl of PDE enzyme in the incubation buffer at 4⁰C
6. Add 5-10 µl of PDE enzyme source (2.5-7.5 ug protein) to above set of tubes and incubate at 4⁰C for 5 minutes.  Care should be taken to pipette the enzyme directly in to the solution.  The supplied partially purified PDE4 enzymes can be used at 5-10 µl/assay. 
7. Make up the volume to 25 µl with fresh incubation buffer.  Keep all tubes at 4⁰C.
8. Prepare appropriate concentration of inhibitor solutions in PDE Substrate Buffer D to get desired concentration in 25 µl.  Up to 5% DMSO can be added without affecting the kit performance.  The effects of other solvents should be tested individually.  
9. Rolipram sensitive PDE4 activity can be determined by adding 25 µl of Rolipram (100 µM in PDE Substrate Buffer D). 
10. Unknown compounds can be screened for PDE4 inhibition by replacing Rolipram with the same volume.
11. The total PDE activity is measured in the absence of any inhibitor.  The inhibitor is replaced by 25 µl of PDE Substrate Buffer D for total PDE activity measurement. 
12. Prepare cAMP buffered substrate fresh for each assay by mixing 100 µl of PDE substrate with 4885 µl of PDE Substrate buffer D and 15 µl of 2, 8 3H-cAMP ammonium salt in ethanol (25-40 Ci/mmole; From Dupont NEN) mix and keep on ice. 
    1. The tube should be marked radioactive and care should be taken for proper handling of radioactive material).
13. Pre-incubate conical tubes with incubation buffer, enzyme source and inhibitors at 30⁰C for 5 minutes.
14. Add 50 µl of buffered PDE substrate (prepared in step 5).
15. Pipette the substrate directly in to the incubation medium and incubate for 10 minutes at 30⁰C with shaking.  It is important to time the addition of substrate as enzymatic reaction is started as soon as substrate is added.  Save the remaining substrate solution for total 3H-CMP counts measurement.  Discard the remaining as liquid radioactive waste.
16. After 10 minutes at 30⁰C, immediately transfer the tubes to a 100⁰C water bath for 3 minutes to stop the PDE reaction.  The tubes may be under pressure due to generation of steam, avoid popping of lids by providing proper ventilation or using tubes with click- lock lids.
17. Immediately transfer the tubes on ice to chill the reaction.  The assay can be stopped at this time and stored at 4⁰C for subsequent steps.

 

Conversion of AMP to Adenosine and separation of adenosine form AMP and cAMP

 

1. Prepare the second enzyme solution be diluting concentrated solution of buffered enzyme 1:10 with distilled water.  Keep at 4⁰C
2. Add 25 µl of enzyme to each tube and incubate at 30⁰C for 15 minutes.  Discard the unused portion, the activity of this enzyme is unstable in dilute solutions. 
3. Add 400 µl of the pre-activated ion exchange resin to each tube.  The resin slurry should be homogeneous during pipetting; this can be achieved by constantly mixing using a small magnetic stirrer bar.
4. Cap and vortex all tubes for 30 seconds. 
5. Incubate all tubes on ice for 15 minutes.
6. Centrifuge all tubes at 13,000 rpm for 2 minutes.  Carefully transfer 150 µl of supernatant for 3H beta liquid scintillation counting.  Keep scintillation vials at room temperature for at least 2 hours before counting.  Discard the remaining incubation mixture in radioactive waste.  The remaining radioactivity will be in the resin pellet.  Handle the remaining tubes as radioactive and discard as solid radioactive waste.
7. Take same amount of radioactive PDE4 substrate for total count measurement.  

 

Calculation of PDE activity

 

The PDE4 activity is calculated by subtracting non-PDE4 activity from Total PDE activity.  The specific activity for PDE4 is defined as µmoles of cAMP hydrolyzed/minute/mg protein. 

 

Trouble shooting

 

• Include at least 2 reagent blanks and 2 denatured enzyme blanks per assay as follows:
  • Prepare 2 tubes with all the reagents except the PDE source, replace PDE with equal volume of Inhibitor Buffer D.  The denatured enzyme blanks will have enzyme source that is denatured at 100⁰C for 3 minutes prior to incubation with substrate. 
  • The reagent blank tubes should not have more than 2-5% of the PDE activity.  If the back ground activity is more, re-centrifuge the tubes and make sure there are no resin beads pipette in the counting solution.
• Rolipram controls are needed for comparison of unknown inhibitors.  Final concentration of Rolipram in the incubation medium is 10 µM to inhibit <95% of the PDE4 activity. 
• Make a quench curve for you scintillation counter for optimal counting efficiency by taking various volumes of incubation supernatants for counting.  If the counts are unstable allow the tubes to sit at room temperature for 2 hours before counting.
• The non-PDE4 activity is measured in the presence of inhibitor Rolipram.  This activity is generally 7-10% of the total activity at 100 µM Rolipram concentration.  Since PDE4 assay is done at 2 µM substrate concentration, if background or non-PDE4 activity is more than 10% of the total activity, the same assay can be performed at 1µM substrate concentration. 

 

Storage Conditions

 

The reagents are stable for 6 months if stored at the indicated temperatures. Please refer to the indicated storage conditions indicated on each reagent bottle or on their specification sheet. 

It is recommended to aliquot the PDE substrate in to 50 µl or 100 µl aliquots to avoid freeze thaw.  Similar aliquots of Buffered Enzyme can be stored at -10⁰C to -20⁰C.  Some precipitation may occur upon storage of buffers at 4⁰C.  Re-dissolve precipitate at room temperature before using these reagents. 

 

General Recommendations

 

We have optimized the reagents for optimal PDE4 activity measurements, however, in some biological samples the relative abundance of non-PDE4 activity may interfere with this assay.  In such situations, the assay can be performed at 1 µM substrate concentration rather than 2 µM.  This will further reduce the contribution of non-PDE4 activity in the total PDE measurements without significantly affecting the PDE4 measurement.  For Ki calculations of PDE4 activity, serially dilute inhibitors in Buffer D such that 25 µl of solution gives desired final inhibitor concentration in incubation medium.