Cardiovascular and Oxidative Stress Assay Kit

NOS Ultrasensitive Colorimetric Assay

$505.00

The Nitric Oxide Synthase Ultrasensitive Colorimetric Assay kit is for the quantitative determination of Nitric Oxide Synthase in biological fluids by a microplate assay. For Research Use Only.

SKU: NSU03-K01 Categories: , ,

NOS Ultrasensitive Colorimetric Assay

The NOS Ultrasensitive Colorimetric Assay is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 1 pmol/µL or ~0.5 µM
Dynamic Range: 0.5 – 100 µM
Incubation Time: 1.5 hours
Sample Type: Cell Lysates
Sample Size: 5 µl
Alternative Name: Nitric Oxide Synthase Ultrasensitive Colorimetric Assay Kit, Ultrasensitive NOS Assay Kit

Product Developed and Manufactured in the USA

Assay Background

The traditional method for measuring nitric oxide synthase (NOS) activity is performed by radiochemical assay that measures the conversion of L-[3H]arginine to L-[3H]citrulline. This method is expensive and requires regulation of radioactive materials. The Ultrasensitive Colorimetric NOS Assay Kit is a low-cost novel assay that allows for the detection of NOS activity without the need for radioactivity. Our Ultrasensitive NOS Assay Kit employs a NADPH recycling system to allow NOS to operate linearly for hours as nitric oxide-derived nitrate and nitrite accumulate. NOS can be assayed spectrophotometrically by measuring the accumulation of its stable degradation products, nitrate and nitrite.  The ratio of these two products in biological fluids, tissue culture media, etc. may vary substantially.  Hence, for accurate assessment of the total nitric oxide generated, one must monitor both nitrate and nitrite.  An excellent solution to this problem is the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase (NaR), followed by quantization of nitrite using Griess Reagent. This kit allows for efficient high-throughout screening of NOS activity in resting cells or cell lysates as well as biological fluids and tissue homogenates. The kit is also ideal for in vitro NOS assays using recombinant purified NOS. All materials necessary to perform the entire assay in a 96-well microplate format are provided with the kit.

Related Products

Nitric Oxide Synthase Colorimetric Assay Kit
Nitric Oxide Synthase Colorimetric Non-Enzymatic Assay Kit
Nitric Oxide Synthase Colorimetric Non-Enzymatic Assay (Refill Kit)

Additional Information

Assay Principle

NADPH and L-arginine are required for the continual operation of NOS and production of nitric oxide (NO). In aqueous solution, NO rapidly degrades to nitrate and nitrite. Spectrophotometric quantization of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs recombinant nitrate reductase (NaR) for conversion of nitrate to nitrite prior to quantization of nitrite using Griess reagent — thus providing for accurate determination of total NOS activity.  This Nitric Oxide Synthase Ultrasensitive Colorimetric Assay kit can be used to accurately measure as little as 1 pmol/µL (~1µM) NO produced in aqueous solutions.  Very little sample is required (5 to 100 µL depending on the [NO] in the sample. The completed reaction is read at 540 nm.

Assay Procedure

In V-well microplate or microcentrifuge tube

  1. Add 40–500 µg of protein from lysates or 0.2 – 1.0 Unit of recombinant or purified NOS in a volume of 30 µl to a tube or well.
  2. Add 200 µL Reaction Buffer
  3. Add 10 µL of NADPH Part A
  4. Add 10 µL of NADPH Part B<
  5. Mix and incubate for 1 – 6 hours at 37°C.
  6. Note: While samples are incubating it is recommended to determine the number of wells to be used and the organization of the samples and standards on the microplate (e.g. see Table 3).
  7. Prepare standards as detailed below and store at 4°C.
  8. Chill on ice for 5 minutes.
  9. Add 10 µL of the reconstituted Nitrate Reductase to each sample, vortex tube or tap plate to mix, and incubate for 20 minutes at room temperature.
  10. Centrifuge at 12,500 rpm for 5 minutes at 4°C.

In flat-bottom microtiter plate

  1. Add, in duplicate, 100 µL of standards to the appropriate wells.
  2. Add, in duplicate, 5-100 µL of sample to the determined wells. The amount of sample per well is dependent upon the amount of NO in the sample.
  3. Add sufficient buffer to each sample to bring the volume to 100 µL. (e.g. 80 µL buffer for 20 µL of sample).
  4. Add 50 µL Color Reagent #1 and shake briefly.
  5. Add 50 µL Color Reagent #2.  Shake for 5 minutes at room temperature.
  6. Read absorbance values at 540 nm in Microtiter plate reader.

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