Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) ELISA Kit (HUFI07499)
- SKU:
- HUFI07499
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q9HC29
- Sensitivity:
- 0.094ng/ml
- Range:
- 0.156-10ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- ACUG, BLAU, CARD15, CD, CLR16.3, IBD1, NLRC2, NOD2, NOD2B, PSORAS1
- Reactivity:
- Human
Description
Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) ELISA
NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) is primarily expressed in the peripheral blood leukocytes. NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) plays a role in the immune response to intracellular bacterial lipopolysaccharides (LPS) by recognizing the muramyl dipeptide (MDP) derived from them and activating the NFKB protein. Mutations in NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) have been associated with Crohns disease and Blau syndrome.
| Product Name: | Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) ELISA Kit |
| Product Code: | HUFI07499 |
| Size: | 96 Assays |
| Alias: | ACUG ELISA Kit, BLAU ELISA Kit, CARD15 ELISA Kit, CD ELISA Kit, CLR16.3 ELISA Kit, IBD1 ELISA Kit, NLRC2 ELISA Kit, NOD2 ELISA Kit, NOD2B ELISA Kit, PSORAS1 ELISA Kit |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) concentrations in serum plasma and other biological fluids. |
| Sensitivity: | < 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) and the recovery rates were calculated by comparing the measured value to the expected amount of Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) in samples. Enquire for more information. |
| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information. |
| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- 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
| UniProt Protein Function: | NOD2: Induces NF-kappa-B via RICK (CARDIAK, RIP2) and IKK- gamma. Confers responsiveness to intracellular bacterial lipopolysaccharides (LPS). Defects in NOD2 are the cause of Blau syndrome (BS). BS is a rare autosomal dominant disorder characterized by early-onset granulomatous arthritis, uveitis and skin rash. Defects in NOD2 are a cause of susceptibility to inflammatory bowel disease type 1 (IBD1). IBD1 is a chronic, relapsing inflammation of the gastrointestinal tract with a complex etiology. It is subdivided into Crohn disease and ulcerative colitis phenotypes. Crohn disease may affect any part of the gastrointestinal tract from the mouth to the anus, but most frequently it involves the terminal ileum and colon. Bowel inflammation is transmural and discontinuous; it may contain granulomas or be associated with intestinal or perianal fistulas. In contrast, in ulcerative colitis, the inflammation is continuous and limited to rectal and colonic mucosal layers; fistulas and granulomas are not observed. Both diseases include extraintestinal inflammation of the skin, eyes, or joints. Defects in NOD2 are the cause of sarcoidosis early-onset (EOS). EOS is a form of sarcoidosis manifesting in children younger than 4 years of age. Sarcoidosis is an idiopathic, systemic, inflammatory disease characterized by the formation of immune granulomas in involved organs. Granulomas predominantly invade the lungs and the lymphatic system, but also skin, liver, spleen, eyes and other organs may be involved. Early- onset sarcoidosis is quite rare and has a distinct triad of skin, joint and eye disorders, without apparent pulmonary involvement. Compared with an asymptomatic and sometimes naturally disappearing course of the disease in older children, early-onset sarcoidosis is progressive and in many cases causes severe complications, such as blindness, joint destruction and visceral involvement. 2 isoforms of the human protein are produced by alternative initiation. |
| UniProt Protein Details: | Chromosomal Location of Human Ortholog: 16q21 Cellular Component: signalosome; cell surface; protein complex; cytoskeleton; cytoplasm; plasma membrane; cytosol; vesicle Molecular Function:protein binding; peptidoglycan binding; enzyme binding; CARD domain binding; protein kinase binding; ATP binding; muramyl dipeptide binding Biological Process: activation of MAPK activity; positive regulation of dendritic cell antigen processing and presentation; maintenance of gastrointestinal epithelium; stress-activated MAPK cascade; response to lipopolysaccharide; toll-like receptor 3 signaling pathway; positive regulation of interleukin-1 beta secretion; positive regulation of interleukin-10 production; activation of NF-kappaB transcription factor; negative regulation of interleukin-2 production; toll-like receptor 5 signaling pathway; positive regulation of gamma-delta T cell activation; positive regulation of phagocytosis; JNK cascade; detection of muramyl dipeptide; cytokine production during immune response; detection of biotic stimulus; toll-like receptor 4 signaling pathway; positive regulation of oxidoreductase activity; positive regulation of interleukin-17 production; negative regulation of interleukin-12 production; positive regulation of T-helper 2 type immune response; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of dendritic cell cytokine production; positive regulation of interleukin-6 production; positive regulation of tumor necrosis factor production; protein oligomerization; negative regulation of interleukin-18 production; toll-like receptor 2 signaling pathway; defense response to Gram-positive bacterium; positive regulation of peptidyl-tyrosine phosphorylation; innate immune response in mucosa; response to muramyl dipeptide; inhibition of NF-kappaB transcription factor; positive regulation of B cell activation; defense response to bacterium; positive regulation of transcription from RNA polymerase II promoter; toll-like receptor 9 signaling pathway; positive regulation of epithelial cell proliferation; negative regulation of T cell mediated immunity; positive regulation of nitric-oxide synthase biosynthetic process; detection of bacterium; positive regulation of interleukin-12 production; positive regulation of JNK cascade; defense response; positive regulation of stress-activated MAPK cascade; toll-like receptor 10 signaling pathway; response to exogenous dsRNA; negative regulation of interferon-gamma production; negative regulation of inflammatory response to antigenic stimulus; positive regulation of biosynthetic process of antibacterial peptides active against Gram-positive bacteria; positive regulation of Notch signaling pathway; response to nutrient; positive regulation of humoral immune response mediated by circulating immunoglobulin; MyD88-independent toll-like receptor signaling pathway; negative regulation of toll-like receptor 2 signaling pathway; positive regulation of phosphoinositide 3-kinase activity; negative regulation of tumor necrosis factor production; immunoglobulin production during immune response; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interleukin-1 beta production; regulation of inflammatory response; toll-like receptor signaling pathway; innate immune response Disease: Psoriatic Arthritis, Susceptibility To; Sarcoidosis, Early-onset; Inflammatory Bowel Disease 1; Blau Syndrome |
| NCBI Summary: | This gene is a member of the Nod1/Apaf-1 family and encodes a protein with two caspase recruitment (CARD) domains and six leucine-rich repeats (LRRs). The protein is primarily expressed in the peripheral blood leukocytes. It plays a role in the immune response to intracellular bacterial lipopolysaccharides (LPS) by recognizing the muramyl dipeptide (MDP) derived from them and activating the NFKB protein. Mutations in this gene have been associated with Crohn disease and Blau syndrome. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. [provided by RefSeq, Jun 2014] |
| UniProt Code: | Q9HC29 |
| NCBI GenInfo Identifier: | 20137973 |
| NCBI Gene ID: | 64127 |
| NCBI Accession: | Q9HC29.1 |
| UniProt Secondary Accession: | Q9HC29,Q96RH5, Q96RH6, Q96RH8, E2JEQ6, |
| UniProt Related Accession: | Q9HC29 |
| Molecular Weight: | 22,403 Da |
| NCBI Full Name: | Nucleotide-binding oligomerization domain-containing protein 2 |
| NCBI Synonym Full Names: | nucleotide-binding oligomerization domain containing 2 |
| NCBI Official Symbol: | NOD2 |
| NCBI Official Synonym Symbols: | CD; ACUG; BLAU; IBD1; NLRC2; NOD2B; CARD15; CLR16.3; PSORAS1 |
| NCBI Protein Information: | nucleotide-binding oligomerization domain-containing protein 2; NOD-like receptor C2; NLR family, CARD domain containing 2; inflammatory bowel disease protein 1; caspase recruitment domain protein 15; nucleotide-binding oligomerization domain 2; caspase recruitment domain family, member 15; caspase recruitment domain-containing protein 15; nucleotide-binding oligomerization domain, leucine rich repeat and CARD domain containing 2 |
| UniProt Protein Name: | Nucleotide-binding oligomerization domain-containing protein 2 |
| UniProt Synonym Protein Names: | Caspase recruitment domain-containing protein 15; Inflammatory bowel disease protein 1 |
| Protein Family: | Nucleotide-binding oligomerization domain-containing protein |
| UniProt Gene Name: | NOD2 |
| UniProt Entry Name: | NOD2_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
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.
| Step | 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. |
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 Type | Protocol |
| 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. |
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