Description
Nitric Oxide Synthase Inhibitor Screening Kit (BA0189) (BA0189)
The Nitric Oxide Synthase Inhibitor Screening Kit (SKU: BA0189) provides a simple, direct and non-radioactive method for screening and evaluating inhibitors of nitric oxide synthase (NOS). Nitric oxide is a reactive radical that plays an important role in many key physiological functions and is the oxidation product of arginine by NOS; inhibition of NOS has the potential to produce diverse biological effects, particularly in the cardiovascular system. The assay involves two steps: a NOS reaction step during which nitric oxide is produced, followed by a detection step in which, because the NO generated is rapidly oxidised to nitrite and nitrate, the NO production is measured following reduction of nitrate to nitrite using an improved Griess method. The colour is read at 540 nm and the homogeneous mix-incubate-measure format can be readily automated, with the whole assay completed in less than 3 hours at 37C. It is intended for research use only.
| Product Name: | Nitric Oxide Synthase Inhibitor Screening Kit (BA0189) |
| SKU: | BA0189 |
| Detection Method: | Colorimetric (OD540 nm, improved Griess method) |
| Sample Type: | Purified NOS with test compounds |
| Species Reactivity: | All |
| Assay Time: | Less than 3 hours (assay performed at 37C) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C (Reagents A, B and C between -20 to 4C) |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A non-radioactive colorimetric assay for screening and evaluating nitric oxide synthase inhibitors. In a two-step procedure, NOS first generates nitric oxide, which is then measured following reduction of nitrate to nitrite using an improved Griess method, with the resulting colour read at 540 nm. The homogeneous format can be completed in less than 3 hours at 37C. Neither NOS enzyme nor a control inhibitor is included in the kit.
- High-throughput homogeneous mix-incubate-measure format readily automated on liquid-handling systems
- Rapid and reliable; can be completed in less than 3 hours when the assay is performed at 37C
- High-throughput screening for NOS inhibitors
- Evaluation of NOS inhibitor potency
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reconstitute Reagent D with 300 uL dH2O, equilibrate all components to room temperature, prewarm Assay Buffer to 25C and keep GDH on ice. Dilute purified NOS to 12.5 U/mL and dissolve test compounds in the solvent of choice (DMSO <= 20 v/v% in the 5 uL added when screening with mouse iNOS). |
| 2 | Transfer 10 uL of NOS into separate wells, reserving at least one well for no substrate (Blank) and one without inhibitor (Control). |
| 3 | Add 5 uL of solvent to the Control and Blank wells and 5 uL of test compound to the remaining NOS wells. Add 25 uL Assay Buffer to all wells and incubate the plate for 15 minutes at 25C. |
| 4 | Prepare Reaction Mix (RM) by mixing, per well: 2 uL Reagent D, 5 uL Reagent E, 5 uL Substrate and 0.5 uL GDH. Prepare Blank Reaction Mix (BRM) with dH2O in place of Substrate. Add 10 uL BRM to the Blank wells and 10 uL RM to the remaining wells, tap to mix and incubate 60 minutes at 37C. |
| 5 | After 60 minutes, prepare NO Detection Reagent by mixing, per well: 100 uL Reagent A, 4 uL Reagent B and 100 uL Reagent C, and immediately add 200 uL to each well. Run the detection reaction at 37C for 60 minutes. |
| 6 | Read OD at 500-570 nm (peak 540 nm). |
% Inhibition = [1 - (delta ODTestCpd / delta ODNoInhibitor)] x 100%, where delta ODTestCpd is the OD540nm of a test compound minus the OD540nm of the Blank well at 60 min, and delta ODNoInhibitor is the OD540nm of the no-inhibitor Control minus the OD540nm of the Blank well at 60 min.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| Substrate | 600 uL | -20C |
| GDH | 120 uL | -20C |
| Reagent A | 12 mL | -20 to 4C |
| Reagent B | 500 uL | -20 to 4C |
| Reagent C | 12 mL | -20 to 4C |
| Reagent D | Dried | -20C |
| Reagent E | 1.5 mL | -20C |
