Neutral Endopeptidase Human Neprilysin Metallo Peptidase Enzymatic LeadHunter Assay - TW

Procedure Summary:

Human CHO cell neutral endopeptidase is used. Test compound and/or vehicle is preincubated with 8.8 ng/ml enzyme* in modified Tris-HCl buffer pH 9.0 for 15 minutes at 25⁰C. The reaction is initiated by addition of 10 μM Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH for another 30 minute incubation period. The plate is read on the spectrophotometer at excitation 320 nm and emission 405 nm. Compounds are screened at 10 μM.

Substrate:

10 µM Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH

Incubation:

30 min at 25°C

Control Inhibitor:

Phosphoramidon

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 µl of 10mM stock -OR- 1 mg (pre-weighed).
2) Dose Response: 60 µl of 10mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2