Overview

  • Product name

    Nitric Oxide Synthase Activity Assay Kit (Fluorometric)
    See all nNOS (neuronal) kits
  • Detection method

    Fluorescent
  • Sample type

    Cell Lysate, Tissue Lysate
  • Assay type

    Enzyme activity (quantitative)
  • Sensitivity

    0.75 µU
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    Nitric Oxide Synthase Activity Assay Kit (Fluorometric) (ab211084) provides an accurate and convenient method to assay nitric oxide synthase (NOS) activity in cell and tissue lysates. In this assay, nitric oxide (NO) generated by NOS undergoes a series of reactions and reacts with the fluorescent probe to generate a stable signal at Ex/Em = 360/450 nm, which is directly proportional to NOS activity. The assay is simple, sensitive and high-throughput adaptable and can detect as low as 0.75 µU of NOS activity.

  • Notes

    Nitric oxide synthases (EC 1.14.13.39) (NOS) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. In presence of NADPH, FAD, FMN, (6R)-5,6,7,8-tetrahydrobiopterin, calmodulin and heme, NOS catalyzes a five-electron oxidation of the guanidino nitrogen of L-arginine with molecular oxygen to generate NO and L-citrulline. There are three isoforms of NOS: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). nNOS accounts for the production of NO in central nervous system, where NO participates in cell communication and information storage. eNOS produces NO in blood vessels and is involved in regulation of vascular function. In contrast to other isoforms, iNOS is expressed de novo under oxidative stress conditions and produces large amounts of NO as a part of body’s defense mechanism.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components Identifier 100 tests
    NOS Assay Buffer WM 1 x 25ml
    Enhancer (0.5 µmole) Purple 1 vial
    NaOH Clear 1 x 1ml
    Nitrate Reductase (1 U) Green 1 vial
    Nitrite Standard (10 µmole) Orange 1 vial
    NOS (Positive Control) Yellow 1 x 4µl
    NOS Cofactor 1 (1 µmole) Blue 1 vial
    NOS Cofactor 2 (25X) Amber 1 x 100µl
    NOS Dilution Buffer Red cap 1 x 1.5ml
    NOS Substrate White 1 x 500µl
    Probe Red/Amber 1 x 1ml
  • Research areas

  • Function

    Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body. In the brain and peripheral nervous system, NO displays many properties of a neurotransmitter. Probably has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such SRR.
  • Tissue specificity

    Isoform 1 is ubiquitously expressed: detected in skeletal muscle and brain, also in testis, lung and kidney, and at low levels in heart, adrenal gland and retina. Not detected in the platelets. Isoform 3 is expressed only in testis. Isoform 4 is detected in testis, skeletal muscle, lung, and kidney, at low levels in the brain, but not in the heart and adrenal gland.
  • Sequence similarities

    Belongs to the NOS family.
    Contains 1 FAD-binding FR-type domain.
    Contains 1 flavodoxin-like domain.
    Contains 1 PDZ (DHR) domain.
  • Domain

    The PDZ domain in the N-terminal part of the neuronal isoform participates in protein-protein interaction, and is responsible for targeting nNos to synaptic membranes in muscles. Mediates interaction with VAC14.
  • Post-translational
    modifications

    Ubiquitinated; mediated by STUB1/CHIP in the presence of Hsp70 and Hsp40 (in vitro).
  • Cellular localization

    Cell membrane > sarcolemma. Cell projection > dendritic spine. In skeletal muscle, it is localized beneath the sarcolemma of fast-twitch muscle fiber by associating with the dystrophin glycoprotein complex. In neurons, enriched in dendritic spines.
  • Information by UniProt
  • Alternative names

    • 2310005C01Rik
    • BNOS
    • Constitutive NOS
    • EC 1.14.13.39
    • IHPS 1
    • IHPS1
    • N-NOS
    • NC-NOS
    • neuronal Nitric Oxide Synthase
    • Neuronal NOS
    • Nitric oxide synthase , neuronal, included
    • Nitric oxide synthase 1
    • Nitric oxide synthase 1 (neuronal)
    • Nitric oxide synthase, brain
    • Nitric oxide synthase, penile neuronal, included
    • NNOS
    • NO
    • NOS
    • NOS 1
    • NOS type I
    • NOS-I
    • NOS1
    • NOS1_HUMAN
    • Peptidyl-cysteine S-nitrosylase NOS1
    see all

Images

  • Nitric Oxide Synthase Activity Assay Kit (Fluorometric) (ab211084). Typical standard calibration curve.

  • Nitric Oxide Synthase Activity Assay Kit (Fluorometric) (ab211084). Detection of endogenous NOS activity in cell lysates from J774.1A mouse monocytes (135 μg) and HeLa cells (157 µg).

  • Nitric Oxide Synthase Activity Assay Kit (Fluorometric) (ab211084). Detection of endogenous NOS activity in J774.1A cell lysates. J774 cells were stimulated with 200 ng/mL LPS and 100 ng/mL murine IFN-gamma. Unstimulated control included. Cell lysates were prepared (225 μg) and assayed following the kit protocol.

  • Nitric Oxide Synthase Activity Assay Kit (Fluorometric) (ab211084). Measurement of NOS Positive Control activity (5 μL) compared to blank (assay buffer).

Protocols

References

ab211084 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Cells overexpressing NOS1

Good Good 4/5 (Ease of Use)
Abreviews
Kit was used with protein extracted from HEK cells transiently expressing the NOS1 wildtype protein as well as cells expressing several NOS1 variants. The amount of protein used was only 5ug, and seemed more than sufficient to perform the assay. However I highlight that 5ug were used in cells overexpressing the NOS1- for cells endogenously expressing NOS1 more tests should be performed to determine the minimum amount of protein necessary.
The standard curve of the assay was perfect. The RFU values of the samples were higher than the ones demonstrated in the examples of the booklet but this can vary according to the reading apparatus. FOr this experiment a SPECTRAMAX apparatus was used to measure the fluorescence by performing a reading from the bottom.
A trial using normal, clear bottom/transparent wells was performed and the results were less accurate so do order the white/clear bottom plates as indicated in the kit guidelines.
Overall the results seem accurate but more trials will be performed in order to verify the reproducibility of the assay.

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Verified customer

Submitted Dec 13 2017

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