Description
Rat Tumor necrosis factor receptor superfamily member 11B (Tnfrsf11b) ELISA Kit
The Rat Tumor Necrosis Factor Receptor Superfamily Member 11B (TNFRSF11B) ELISA Kit is a highly sensitive and specific assay designed for the accurate quantification of TNFRSF11B levels in rat serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results, making it suitable for a wide range of research applications.TNFRSF11B, also known as Osteoprotegerin (OPG), is a key regulator of bone metabolism and plays a crucial role in bone remodeling. It acts as a decoy receptor for RANKL, inhibiting osteoclast differentiation and activation.
Dysregulation of TNFRSF11B has been implicated in various bone disorders, including osteoporosis and rheumatoid arthritis, making it a valuable biomarker for studying these conditions and developing potential therapeutic interventions.The Rat TNFRSF11B ELISA Kit offers researchers a powerful tool for studying the role of TNFRSF11B in bone metabolism and related diseases, with its high precision and accuracy ensuring reliable results for their experiments.
| Product Name: | Rat Tumor necrosis factor receptor superfamily member 11B (Tnfrsf11b) ELISA Kit |
| SKU: | RTEB0084 |
| Size: | 96T |
| Target: | Rat Tumor necrosis factor receptor superfamily member 11B (Tnfrsf11b) |
| Synonyms: | Osteoprotegerin, Opg |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Rat |
| Detection Range: | 15.6-1000pg/mL |
| Sensitivity: | 7.8pg/mL |
| Intra CV: | 4.8% | ||||||||||||||||||||
| Inter CV: | 7.7% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Acts as decoy receptor for TNFSF11/RANKL and thereby neutralizes its function in osteoclastogenesis. Inhibits the activation of osteoclasts and promotes osteoclast apoptosis in vitro. Bone homeostasis seems to depend on the local ratio between TNFSF11 and TNFRSF11B. May also play a role in preventing arterial calcification. May act as decoy receptor for TNFSF10/TRAIL and protect against apoptosis. TNFSF10/TRAIL binding blocks the inhibition of osteoclastogenesis. |
| Uniprot: | O08727 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant rat Tumor necrosis factor receptor superfamily member 11B |
| Sub Unit: | Homodimer. Interacts with TNFSF10 and TNFSF11. |
| Research Area: | Immunology |
| Subcellular Location: | Secreted |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | TNFRSF11B: Acts as decoy receptor for RANKL and thereby neutralizes its function in osteoclastogenesis. Inhibits the activation of osteoclasts and promotes osteoclast apoptosis in vitro. Bone homeostasis seems to depend on the local RANKL/OPG ratio. May also play a role in preventing arterial calcification. May act as decoy receptor for TRAIL and protect against apoptosis. TRAIL binding blocks the inhibition of osteoclastogenesis. Defects in TNFRSF11B are the cause of juvenile Paget disease (JPD); also known as hyperostosis corticalis deformans juvenilis or hereditary hyperphosphatasia or chronic congenital idiopathic hyperphosphatasia. JPD is a rare autosomal recessive osteopathy that presents in infancy or early childhood. The disorder is characterized by rapidly remodeling woven bone, osteopenia, debilitating fractures, and deformities due to a markedly accelerated rate of bone remodeling throughout the skeleton. Approximately 40 cases of JPD have been reported worldwide. Unless it is treated with drugs that block osteoclast- mediated skeletal resorption, the disease can be fatal. |
| UniProt Protein Details: | Protein type:Secreted; Inhibitor; Secreted, signal peptide Chromosomal Location of Human Ortholog: 8q24 Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region Molecular Function:cytokine activity; receptor activity Biological Process: response to drug; response to magnesium ion; extracellular matrix organization and biogenesis; negative regulation of odontogenesis of dentine-containing teeth; response to estrogen stimulus; apoptosis; response to arsenic; signal transduction; negative regulation of bone resorption; skeletal development; response to nutrient Disease: Paget Disease, Juvenile |
| NCBI Summary: | The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein is an osteoblast-secreted decoy receptor that functions as a negative regulator of bone resorption. This protein specifically binds to its ligand, osteoprotegerin ligand, both of which are key extracellular regulators of osteoclast development. Studies of the mouse counterpart also suggest that this protein and its ligand play a role in lymph-node organogenesis and vascular calcification. Alternatively spliced transcript variants of this gene have been reported, but their full length nature has not been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O08727 |
| NCBI GenInfo Identifier: | 148743793 |
| NCBI Gene ID: | 4982 |
| NCBI Accession: | NP_002537.3 |
| UniProt Secondary Accession: | O08727,O08712, O08727, |
| UniProt Related Accession: | O00300 |
| Molecular Weight: | |
| NCBI Full Name: | tumor necrosis factor receptor superfamily member 11B |
| NCBI Synonym Full Names: | TNF receptor superfamily member 11b |
| NCBI Official Symbol: | TNFRSF11B |
| NCBI Official Synonym Symbols: | OPG; TR1; OCIF; PDB5 |
| NCBI Protein Information: | tumor necrosis factor receptor superfamily member 11B |
| UniProt Protein Name: | Tumor necrosis factor receptor superfamily member 11B |
| UniProt Synonym Protein Names: | Osteoclastogenesis inhibitory factor; Osteoprotegerin |
| UniProt Gene Name: | TNFRSF11B |
| UniProt Entry Name: | TR11B_HUMAN |
| 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. |
