Description
NOS Activity Assay Kit (Colorimetric) (BA0138) (BA0138)
The NOS Activity Assay Kit (Colorimetric) (SKU: BA0138) provides a simple, non-radioactive method for measuring nitric oxide synthase (NOS) activity in biological samples. Nitric oxide (NO) is a reactive radical that plays an important role in many physiological functions, being produced by the oxidation of arginine by NOS and involved in host defence, development, activation of regulatory proteins and covalent interaction with functional biomolecules. This assay involves two steps: a NOS reaction step during which NO is produced, followed by an NO detection step. Since the NO generated is rapidly oxidised to nitrite and nitrate, NO production is measured following reduction of nitrate to nitrite using an improved Griess method, with the reaction product read at a peak of 540 nm. The procedure is simple and the time required for sample pretreatment and assay is reduced to as short as 40 minutes. The detection range is 0.25 to 25 U/L in a 96-well plate.
| Product Name: | NOS Activity Assay Kit (Colorimetric) (BA0138) |
| SKU: | BA0138 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.25 to 25 U/L |
| Sample Type: | Tissue, cell samples, serum, plasma, whole blood and cell culture media |
| Species Reactivity: | All |
| Assay Time: | As short as 40 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20 degrees C (Assay Buffer, Substrate, Reagent D, Reagent E and GDH at -20 degrees C; all other reagents at -20 to 4 degrees C) |
| Shelf Life: | 6 months after receipt (use Reagent D within 1 week after reconstitution) |
| Shipping: | Gel Pack |
This kit involves two steps: a NOS reaction step during which NO is produced, followed by an NO detection step. Because the NO generated by NOS is rapidly oxidised to nitrite and nitrate, NO production is measured following reduction of nitrate to nitrite using an improved Griess method, and the reaction product is read at a peak of 540 nm.
- Sensitive and accurate with a detection range of 0.25 to 25 U/L in a 96-well plate
- Rapid and reliable, and can be completed in 40 minutes if reduction of nitrate to nitrite is performed at 60 degrees C
- Simple, direct and non-radioactive procedure
- Direct measurement of NOS activity in biological samples
- Drug discovery and pharmacology studies of the effects of drugs on NOS activity
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 | Equilibrate all components to room temperature, reconstitute one tube of Reagent D with 300 uL distilled water (reconstitute both tubes if assaying more than 60 wells), prewarm Assay Buffer to 37 degrees C, keep GDH on ice, and warm Reagent B at 37 degrees C if precipitates are present. |
| 2 | Prepare 200 uL 500 uM Premix by mixing 100 uL 1.0 mM Standard and 100 uL distilled water, then dilute the standards as described in the dilution table. |
| 3 | Add 20 uL of each sample and standard to separate labelled Eppendorf tubes (two tubes per sample: one reaction tube and one sample blank), prepare NOS Working Reagent by mixing per reaction tube 65 uL Assay Buffer, 4 uL Substrate, 4 uL reconstituted Reagent D, 10 uL Reagent E and 1 uL GDH (for the sample blanks use 8 uL distilled water instead of the Substrate and Reagent D), add 80 uL to each tube and incubate at 37 degrees C for 20 minutes. |
| 4 | For samples requiring deproteination, add 7 uL ZnSO4 to each sample and standard tube, vortex, add 7 uL NaOH, vortex again, centrifuge 10 minutes at 14,000 rpm and transfer 100 uL of the clear supernatant to a clean tube. |
| 5 | Prepare NO Detection Reagent by mixing per reaction tube 100 uL Reagent A, 4 uL Reagent B and 100 uL Reagent C, add 200 uL to each sample and standard tube and incubate for 5 minutes at 60 degrees C (alternatively 37 degrees C for 60 minutes or room temperature for 150 minutes). |
| 6 | Briefly centrifuge the reaction tubes and transfer 250 uL of each reaction to separate wells in a 96-well plate, then read OD at 500-570 nm (peak 540 nm). |
Subtract the blank OD (Std 4) from the standard OD values and plot the OD against standard concentrations, determining the slope by linear regression. NOS Activity = (ODSAMPLE - ODBLANK) / Slope x (1/t) (U/L), where t is the reaction time (20 minutes). One unit of NOS catalyses the production of 1 umole of nitric oxide per minute under the assay conditions (pH 7.5 and 37 degrees C).
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 degrees C |
| Substrate | 600 uL | -20 degrees C |
| GDH | 120 uL | -20 degrees C |
| Reagent A | 12 mL | -20 to 4 degrees C |
| Reagent B | 500 uL | -20 to 4 degrees C |
| Reagent C | 12 mL | -20 to 4 degrees C |
| Reagent D (Dried) | 2 Tubes | -20 degrees C |
| Reagent E | 1.5 mL | -20 degrees C |
| ZnSO4 | 1 mL | -20 to 4 degrees C |
| Standard | 1 mL | -20 to 4 degrees C |
| NaOH | 1 mL | -20 to 4 degrees C |
