Mouse Cell Signalling ELISA Kits 1
Mouse Tumor necrosis factor (Tnf) ELISA Kit
- SKU:
- MOEB0127
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P06804
- Range:
- 7.8-500 pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- TNFalpha, Tumor Necrosis Factor Alpha, TNF-alpha, DIF, TNF-alpha, TNFA, TNFSF2
- Reactivity:
- Mouse
Description
| Product Name: | Mouse Tumor necrosis factor (Tnf) ELISA Kit |
| Product Code: | MOEB0127 |
| Alias: | Tumor necrosis factor, Cachectin, TNF-alpha, Tumor necrosis factor ligand superfamily member 2, TNF-a, Tnf, Tnfa, Tnfsf2 |
| Uniprot: | P06804 |
| Reactivity: | Mouse |
| Range: | 7.8-500 pg/mL |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | TNF-a: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Homotrimer. Interacts with SPPL2B. Belongs to the tumor necrosis factor family. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Membrane protein, integral; Apoptosis; Cytokine Cellular Component: extracellular space; cell surface; integral to plasma membrane; extracellular region; integral to membrane; lipid raft; secretory granule; recycling endosome; membrane; plasma membrane; intracellular; phagocytic cup; external side of plasma membrane Molecular Function:identical protein binding; protein binding; protease binding; cytokine activity; tumor necrosis factor receptor binding Biological Process: positive regulation of JNK activity; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; positive regulation of NFAT protein import into nucleus; activation of MAPK activity; positive regulation of osteoclast differentiation; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; multicellular organismal development; response to glucocorticoid stimulus; positive regulation of caspase activity; positive regulation of NF-kappaB import into nucleus; osteoclast differentiation; positive regulation of translational initiation by iron; positive regulation of membrane protein ectodomain proteolysis; activation of NF-kappaB transcription factor; positive regulation of MAP kinase activity; tumor necrosis factor-mediated signaling pathway; cellular extravasation; positive regulation of phagocytosis; negative regulation of interleukin-6 production; JNK cascade; negative regulation of osteoblast differentiation; positive regulation of action potential; regulation of immunoglobulin secretion; negative regulation of protein complex disassembly; positive regulation of cytokine production; positive regulation of heterotypic cell-cell adhesion; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of mitosis; response to virus; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; glucose metabolic process; positive regulation of chemokine production; negative regulation of cytokine secretion during immune response; positive regulation of protein transport; detection of mechanical stimulus involved in sensory perception of pain; cell activation; defense response to Gram-positive bacterium; organ morphogenesis; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; defense response to bacterium; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; negative regulation of L-glutamate transport; negative regulation of transcription, DNA-dependent; leukocyte migration; sequestering of triacylglycerol; apoptosis; positive regulation of smooth muscle cell proliferation; positive regulation of JNK cascade; defense response; negative regulation of transcription from RNA polymerase II promoter; positive regulation of interleukin-18 production; signal transduction; chronic inflammatory response to antigenic stimulus; positive regulation of synaptic transmission; positive regulation of hair follicle development; negative regulation of cell proliferation; regulation of protein secretion; regulation of osteoclast differentiation; negative regulation of lipid catabolic process; positive regulation of neuron apoptosis; positive regulation of cell proliferation; lipopolysaccharide-mediated signaling pathway; protein kinase B signaling cascade; positive regulation of chronic inflammatory response to antigenic stimulus; regulation of I-kappaB kinase/NF-kappaB cascade; inflammatory response; caspase activation; positive regulation of humoral immune response mediated by circulating immunoglobulin; regulation of protein amino acid phosphorylation; positive regulation of protein complex disassembly; transformed cell apoptosis; calcium-mediated signaling; MAPKKK cascade; positive regulation of peptidyl-serine phosphorylation; humoral immune response; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; cell proliferation; positive regulation of interferon-gamma production; negative regulation of glucose import; positive regulation of programmed cell death; positive regulation of chemokine biosynthetic process; positive regulation of protein complex assembly; negative regulation of viral genome replication; protein import into nucleus, translocation; positive regulation of protein kinase activity; activation of MAPKKK activity; positive regulation of fever; immune response; positive regulation of protein amino acid phosphorylation; receptor biosynthetic process; negative regulation of myoblast differentiation; leukocyte tethering or rolling; regulation of insulin secretion; positive regulation of cytokine secretion; positive regulation of inflammatory response |
| NCBI Summary: | This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. Members of this family are classified based on primary sequence, function, and structure. This protein is synthesized as a type-II transmembrane protein and is reported to be cleaved into products that exert distinct biological functions. It plays an important role in the innate immune response as well as regulating homeostasis but is also implicated in diseases of chronic inflammation. In mouse deficiency of this gene is associated with defects in response to bacterial infection, with defects in forming organized follicular dendritic cell networks and germinal centers, and with a lack of primary B cell follicles. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jun 2013] |
| UniProt Code: | P06804 |
| NCBI GenInfo Identifier: | 135935 |
| NCBI Gene ID: | 21926 |
| NCBI Accession: | P06804.2 |
| UniProt Secondary Accession: | P06804,O35853, Q62326, Q91VF3, |
| UniProt Related Accession: | P06804 |
| Molecular Weight: | 25,896 Da |
| NCBI Full Name: | Tumor necrosis factor |
| NCBI Synonym Full Names: | tumor necrosis factor |
| NCBI Official Symbol: | Tnf  |
| NCBI Official Synonym Symbols: | DIF; Tnfa; TNF-a; TNFSF2; Tnfsf1a; TNFalpha; TNF-alpha  |
| NCBI Protein Information: | tumor necrosis factor; cachectin; tumor necrosis factor-alpha; tumor necrosis factor ligand superfamily member 2 |
| UniProt Protein Name: | Tumor necrosis factor |
| UniProt Synonym Protein Names: | Cachectin; TNF-alpha; Tumor necrosis factor ligand superfamily member 2; TNF-a |
| Protein Family: | Tumor necrosis factor |
| UniProt Gene Name: | Tnf  |
| UniProt Entry Name: | TNFA_MOUSE |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 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
*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.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
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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