Human iNOS / Nitric oxide synthase, inducible ELISA Kit

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SKU:
HUFI00403
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human iNOS / Nitric oxide synthase, inducible ELISA Kit - Information

The ELISA Genie iNOS / Nitric oxide synthase, inducible ELISA Kit can assay for iNOS / Nitric oxide synthase, inducible in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our iNOS / Nitric oxide synthase, inducible ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound iNOS / Nitric oxide synthase, inducible is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human iNOS / Nitric oxide synthase, inducible ELISA Kit - Data

Description

Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body (PubMed:7531687, PubMed:7544004). In macrophages, NO mediates tumoricidal and bactericidal actions. Also has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such PTGS2/COX2. As component of the iNOS-S100A8/9 transnitrosylase complex involved in the selective inflammatory stimulus-dependent S-nitrosylation of GAPDH on 'Cys-247' implicated in regulation of the GAIT complex activity and probably multiple targets including ANXA5, EZR, MSN and VIM (PubMed:25417112). Involved in inflammation, enhances the synthesis of proinflammatory mediators such as IL6 and IL8 (PubMed:19688109).

Post-Translational Modification

Uniprot ID P35228
Alias

NOS2/iNOS/NOS2A/HEP-NOS/I-NOS/Hepatocyte NOS/Peptidyl-cysteine S-nitrosylase NOS2/Inducible NO synthase/Inducible NOS/INOS/NOS/NOS type II/NOS type II

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of NOS2/iNOS concentrations in serum plasma and other biological fluids.

Size

96T

Range

78.125-5000pg/ml

Sensitivity

< 46.875pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of NOS2/iNOS and the recovery rates were calculated by comparing the measured value to the expected amount of NOS2/iNOS in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-102 95
EDTA plasma(n=5) 87-97 91
UFH plasma(n=5) 85-101 93
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of NOS2/iNOS and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 85-101% 87-97% 92-101% 93-105%
EDTA plasma(n=5) 84-101% 86-99% 84-93% 82-95%
UFH plasma(n=5) 86-100% 80-96% 80-97% 85-94%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human iNOS / Nitric oxide synthase, inducible ELISA Kit Protocol

The below protocol is a sample protocol for Human iNOS / Nitric oxide synthase, inducible ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human iNOS / Nitric oxide synthase, inducible present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Human iNOS / Nitric oxide synthase, inducible ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human iNOS / Nitric oxide synthase, inducible ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Human iNOS / Nitric oxide synthase, inducible ELISA Kit Protein Information

UniProt Protein Function:Function: Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body. In macrophages, NO mediates tumoricidal and bactericidal actions. Also has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such COX2.
UniProt Protein Details:

Catalytic activity: 2 L-arginine + 3 NADPH + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O.

Cofactor: Heme group

By similarity.Binds 1 FAD

By similarity.Binds 1 FMN

By similarity.Tetrahydrobiopterin (BH4). May stabilize the dimeric form of the enzyme

By similarity.

Enzyme regulation: Regulated by calcium/calmodulin. Aspirin inhibits expression and function of this enzyme and effects may be exerted at the level of translational/post-translational modification and directly on the catalytic activity

By similarity.

Subunit structure: Homodimer. Binds SLC9A3R1.

Tissue specificity: Expressed in the liver, retina, bone cells and airway epithelial cells of the lung. Not expressed in the platelets.

Induction: By endotoxins and cytokines. Induced by IFNG/IFN-gamma acting synergistically with bacterial lipopolysaccharides (LPS), TNF or IL1B/interleukin-1 beta. Ref.14

Polymorphism: Note=Genetic variations in NOS2 are involved in resistance to malaria [

MIM:611162].

Sequence similarities: Belongs to the NOS family.Contains 1 FAD-binding FR-type domain.Contains 1 flavodoxin-like domain.

NCBI Summary:Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. This gene encodes a nitric oxide synthase which is expressed in liver and is inducible by a combination of lipopolysaccharide and certain cytokines. Three related pseudogenes are located within the Smith-Magenis syndrome region on chromosome 17. [provided by RefSeq, Jul 2008]
UniProt Code:P35228
NCBI GenInfo Identifier:1352513
NCBI Gene ID:4843
NCBI Accession:P35228.2
UniProt Secondary Accession:P35228,O60757, O94994, Q16263, Q16692, Q4TTS5, Q9UD42 A1L3U5, B7ZLY2,
UniProt Related Accession:P35228
Molecular Weight:131,117 Da
NCBI Full Name:Nitric oxide synthase, inducible
NCBI Synonym Full Names:nitric oxide synthase 2, inducible
NCBI Official Symbol:NOS2  
NCBI Official Synonym Symbols:NOS; INOS; NOS2A; HEP-NOS  
NCBI Protein Information:nitric oxide synthase, inducible; NOS type II; NOS, type II; inducible NOS; hepatocyte NOS; inducible NO synthase; nitric oxide synthase, macrophage; peptidyl-cysteine S-nitrosylase NOS2; nitric oxide synthase 2A (inducible, hepatocytes)
UniProt Protein Name:Nitric oxide synthase, inducible
UniProt Synonym Protein Names:Hepatocyte NOS; HEP-NOS; Inducible NO synthase; Inducible NOS; iNOS; NOS type II; Peptidyl-cysteine S-nitrosylase NOS2
Protein Family:Inositol 3-kinase
UniProt Gene Name:NOS2  
UniProt Entry Name:NOS2_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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