[3H] Phosphodiesterase enzyme assay
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384-well assay protocol>
Reagent usage in 96-, 384-well formats>
Typical data>
Assay concept
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384-well assay protocol
- 2.5 µl assay buffer (500 mM Tris, pH 7.5, 83 mM MgCl2, 17 mM EGTA)
- 2.5 µl [3H]cAMP (TRK559)
- 2.5 µl PDE (0.156 mg)
- Add 12.5 µl de-ionised water
- Incubate for 30 minutes at 37oC
- Add 15 µl yttrium oxide membrane binding beads (RPNQ0280 0.3 mg) in 40 mM ZnSO4
- Shake for 2 minutes
- Allow beads to settle for 20 minutes
- Image for 10 minutes
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Reagent usage in 96- and 384-well format
Assay components | 96-well | 384-well |
Assay volume | 150 µl | 35 µl |
Bead | 1 µg | 0.3 µg |
Enzyme | 0.5 µg | 0.156 µg |
[3H]cAMP | .05 µCi | 0.0125 µCi |
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Typical data
Enzyme titration in 384-well microplate
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