Description
Rat Mothers against decapentaplegic homolog 2 (Smad2) ELISA Kit
The Rat Mothers Against Decapentaplegic Homolog 2 (SMAD2) ELISA Kit is specifically designed for the accurate measurement of SMAD2 levels in rat serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.SMAD2 is a key protein involved in the TGF-β signaling pathway, regulating cellular processes such as cell growth, differentiation, and apoptosis.
Dysregulation of SMAD2 has been implicated in various diseases, including cancer, fibrosis, and developmental disorders, highlighting its importance as a potential biomarker for disease progression and therapeutic development.With this ELISA Kit, researchers can confidently measure SMAD2 levels in rat samples, allowing for a better understanding of its role in disease pathogenesis and providing valuable insights for future therapeutic interventions.
| Product Name: | Rat Mothers against decapentaplegic homolog 2 (Smad2) ELISA Kit |
| SKU: | RTEB1417 |
| Size: | 96T |
| Target: | Rat Mothers against decapentaplegic homolog 2 (Smad2) |
| Synonyms: | Mad-related protein 2, SMAD family member 2, SMAD 2, MAD homolog 2, Madh2 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Rat |
| Detection Range: | 0.625-40ng/mL |
| Sensitivity: | 0.316ng/mL |
| Intra CV: | 4.9% | ||||||||||||||||||||
| Inter CV: | 8.4% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD2/SMAD4 complex, activates transcription. May act as a tumor suppressor in colorectal carcinoma. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator. |
| Uniprot: | O70436 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant rat Mothers against decapentaplegic homolog 2 |
| Sub Unit: | Monomer; the absence of TGF-beta. Interacts with ZNF580. Heterodimer; in the presence of TGF-beta. Forms a heterodimer with co-SMAD, SMAD4, in the nucleus to form the transactivation complex SMAD2/SMAD4. Found in a complex with ACVR1B, SMAD3 and TRIM33 upon addition of TGF-beta. Interacts with SMAD3 and TRIM33. Interacts (via the MH2 domain) with ZFYVE9; may form trimers with the SMAD4 co-SMAD. Interacts with FOXH1, homeobox protein TGIF, PEBP2-alpha subunit, CREB-binding protein (CBP), EP300, SKI and SNW1. Interacts with SNON; when phosphorylated at Ser-465/467. Interacts (via PY-motif) with SMURF2. Interacts with AIP1 and HGS. Interacts with NEDD4L in response to TGF-beta. Interacts with SKOR1 and SKOR2. Interacts with PRDM16. Interacts (via MH2 domain) with LEMD3. Interacts with RBPMS. Interacts with WWP1. Interacts (dephosphorylated form, via the MH1 and MH2 domains) with RANBP3 (via its C-terminal R domain); the interaction results in the export of dephosphorylated SMAD3 out of the nucleus and termination of the TGF-beta signaling. Interacts with PML. Interacts with PDPK1 (via PH domain). Interacts with DAB2; the interactions are enhanced upon TGF-beta stimulation. Interacts with USP15. Interacts with PPP5C. Interacts with LDLRAD4 (via the SMAD interaction motif). Interacts (via MH2 domain) with PMEPA1 (via the SMAD interaction motif). Interacts with ZFHX3. Interacts with ZNF451. Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts weakly with ZNF8. Interacts (when phosphorylated) with RNF111; RNF111 acts as an enhancer of the transcriptional responses by mediating ubiquitination and degradation of SMAD2 inhibitors. |
| Subcellular Location: | Cytoplasm Nucleus Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | SMAD2: ubiquitously expressed transcription factor phosphorylated and activated by TGF-beta receptor-type kinases. Participates in a wide range of critical processes including morphogenesis, cell-fate determination, proliferation, differentiation and apoptosis. Phosphorylated Smads dimerize with collaborating Smad4 and are translocated into the nucleus, where the transcription of target genes is stimulated. Two alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 18q21.1 Cellular Component: cytoplasm; cytosol; nuclear chromatin; nucleoplasm; nucleus; transcription factor complex Molecular Function:chromatin binding; DNA binding; double-stranded DNA binding; metal ion binding; phosphatase binding; protein binding; protein heterodimerization activity; protein homodimerization activity; SMAD binding; transcription activator binding; transcription factor activity; transcription factor binding; transforming growth factor beta receptor binding; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity; ubiquitin protein ligase binding Biological Process: activin receptor signaling pathway; anterior/posterior pattern formation; cell fate commitment; common-partner SMAD protein phosphorylation; embryonic cranial skeleton morphogenesis; embryonic foregut morphogenesis; endoderm formation; gastrulation; gene expression; in utero embryonic development; insulin secretion; lung development; mesoderm formation; negative regulation of cell proliferation; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; negative regulation of transforming growth factor beta receptor signaling pathway; organ growth; palate development; pancreas development; paraxial mesoderm morphogenesis; pericardium development; positive regulation of BMP signaling pathway; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; post-embryonic development; primary microRNA processing; regulation of binding; regulation of transforming growth factor beta receptor signaling pathway; response to glucose stimulus; SMAD protein complex assembly; somatic stem cell maintenance; transcription initiation from RNA polymerase II promoter; transcription, DNA-dependent; transforming growth factor beta receptor signaling pathway; ureteric bud development; zygotic determination of dorsal/ventral axis |
| NCBI Summary: | The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to TGF-beta signal, this protein is phosphorylated by the TGF-beta receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The association with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, May 2012] |
| UniProt Code: | O70436 |
| NCBI GenInfo Identifier: | 13633914 |
| NCBI Gene ID: | 4087 |
| NCBI Accession: | Q15796.1 |
| UniProt Secondary Accession: | O70436,Q62432, O70436, |
| UniProt Related Accession: | Q15796 |
| Molecular Weight: | 48,956 Da |
| NCBI Full Name: | Mothers against decapentaplegic homolog 2 |
| NCBI Synonym Full Names: | SMAD family member 2 |
| NCBI Official Symbol: | SMAD2 |
| NCBI Official Synonym Symbols: | JV18; MADH2; MADR2; JV18-1; hMAD-2; hSMAD2 |
| NCBI Protein Information: | mothers against decapentaplegic homolog 2 |
| UniProt Protein Name: | Mothers against decapentaplegic homolog 2 |
| UniProt Synonym Protein Names: | JV18-1; Mad-related protein 2; hMAD-2; SMAD family member 2; SMAD 2; Smad2; hSMAD2 |
| Protein Family: | Mothers against decapentaplegic |
| UniProt Gene Name: | SMAD2 |
| UniProt Entry Name: | SMAD2_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. |
