Description
Rat Mothers against decapentaplegic homolog 3 (Smad3) ELISA Kit
The Rat Mothers Against Decapentaplegic Homolog 3 (SMAD3) ELISA Kit is a powerful tool for the quantitative measurement of SMAD3 levels in rat biological samples. This kit boasts high sensitivity and specificity, ensuring accurate and reliable results for various research applications.SMAD3 is a key signaling molecule in the TGF-beta pathway, playing a critical role in regulating cell growth, differentiation, and apoptosis. Dysregulation of SMAD3 has been linked to numerous diseases, including fibrosis, cancer, and inflammatory disorders, highlighting its importance as a potential therapeutic target and biomarker.
With the Rat SMAD3 ELISA Kit, researchers can explore the role of SMAD3 in disease pathogenesis, drug development, and biomarker discovery, advancing our understanding of complex biological processes and paving the way for new treatment strategies.
| Product Name: | Rat Mothers against decapentaplegic homolog 3 (Smad3) ELISA Kit |
| SKU: | RTEB1418 |
| Size: | 96T |
| Target: | Rat Mothers against decapentaplegic homolog 3 (Smad3) |
| Synonyms: | SMAD family member 3, SMAD 3, MAD homolog 3, Madh3 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Rat |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.196ng/mL |
| Intra CV: | Provided with the Kit |
| Inter CV: | Provided with the Kit |
| Linearity: | Provided with the Kit |
| Recovery: | Provided with the Kit |
| 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 SMAD3/SMAD4 complex, activates transcription. Also can form a SMAD3/SMAD4/JUN/FOS complex at the AP-1/SMAD site to regulate TGF-beta-mediated transcription. Has an inhibitory effect on wound healing probably by modulating both growth and migration of primary keratinocytes and by altering the TGF-mediated chemotaxis of monocytes. This effect on wound healing appears to be hormone-sensitive. Regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator. |
| Uniprot: | P84025 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant rat Mothers against decapentaplegic homolog 3 |
| Sub Unit: | Monomer; in the absence of TGF-beta. Homooligomer; in the presence of TGF-beta. Heterotrimer; forms a heterotrimer in the presence of TGF-beta consisting of two molecules of C-terminally phosphorylated SMAD2 or SMAD3 and one of SMAD4 to form the transcriptionally active SMAD2/SMAD3-SMAD4 complex. Interacts with TGFBR1. Interaction with CSNK1G2. Interacts (via the MH2 domain) with ZFYVE9. Interacts with HDAC1, VDR, TGIF and TGIF2, RUNX3, CREBBP, SKOR1, SKOR2, SNON, ATF2, SMURF2 and SNW1. Interacts with DACH1; the interaction inhibits the TGF-beta signaling. Part of a complex consisting of AIP1, ACVR2A, ACVR1B and SMAD3. Forms a complex with SMAD2 and TRIM33 upon addition of TGF-beta. Found in a complex with SMAD3, RAN and XPO4. Interacts in the complex directly with XPO4. Interacts (via the MH2 domain) with LEMD3; the interaction represses SMAD3 transcriptional activity through preventing the formation of the heteromeric complex with SMAD4 and translocation to the nucleus. Interacts with RBPMS. Interacts (via MH2 domain) with MECOM. Interacts with WWTR1 (via its coiled-coil domain). Interacts (via the linker region) with EP300 (C-terminal); the interaction promotes SMAD3 acetylation and is enhanced by TGF-beta phosphorylation in the C-terminal of SMAD3. This interaction can be blocked by competitive binding of adenovirus oncoprotein E1A to the same C-terminal site on EP300, which then results in partially inhibited SMAD3/SMAD4 transcriptional activity. Interacts with SKI; the interaction represses SMAD3 transcriptional activity. Component of the multimeric complex SMAD3/SMAD4/JUN/FOS which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. Interacts (via an N-terminal domain) with JUN (via its basic DNA binding and leucine zipper domains); this interaction is essential for DNA binding and cooperative transcriptional activity in response to TGF-beta. Interacts with PPM1A; the interaction dephosphorylates SMAD3 in the C-terminal SXS motif leading to disruption of the SMAD2/3-SMAD4 complex, nuclear export and termination of TGF-beta signaling. 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 AIP1, PML, TGFB1I1, TTRAP, FOXL2, PRDM16, HGS and WWP1. Interacts with NEDD4L; the interaction requires TGF-beta stimulation. Interacts with MEN1. Interacts (via MH2 domain) with CITED2 (via C-terminus). Interacts with PDPK1 (via PH domain). Interacts with DAB2; the interactions are enhanced upon TGF-beta stimulation. Interacts with USP15. Interacts with PPP5C; the interaction decreases SMAD3 phosphorylation and protein levels. Interacts with LDLRAD4 (via the SMAD interaction motif). Interacts with PMEPA1. Interacts with ZC3H3 (By similarity). Interacts with ZFHX3 (By similarity). Interacts with ZNF451. Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4 (By similarity). Interacts weakly with ZNF8. Interacts with STUB1, HSPA1A, HSPA1B, HSP90AA1 and HSP90AB1. |
| Research Area: | Cancer |
| 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. Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. Co-localizes with LEMD3 at the nucleus inner membrane. MAPK-mediated phosphorylation appears to have no effect on nuclear import. PDPK1 prevents its nuclear translocation in response to TGF-beta. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | SMAD3: transcription factor phosphorylated and activated by TGF-beta-type receptors. A receptor-regulated Smad (R-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. In mouse required for establishemnt of the mucosal immune response and proper development of skeleton. |
| UniProt Protein Details: | Protein type:DNA-binding; Nuclear receptor co-regulator; Transcription factor Chromosomal Location of Human Ortholog: 8q24 Cellular Component: cytoplasm; nuclear chromatin; nuclear inner membrane; nucleoplasm; nucleus; plasma membrane; protein complex; receptor complex; transcription factor complex Molecular Function:beta-catenin binding; bHLH transcription factor binding; chromatin binding; chromatin DNA binding; collagen binding; DNA binding; double-stranded DNA binding; enzyme binding; identical protein binding; phosphatase binding; protein binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; sequence-specific DNA binding; SMAD binding; transcription factor activity; transcription factor binding; transforming growth factor beta receptor binding; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity; ubiquitin binding; ubiquitin protein ligase binding; zinc ion binding Biological Process: activin receptor signaling pathway; caspase activation; cell cycle arrest; developmental growth; embryonic cranial skeleton morphogenesis; embryonic foregut morphogenesis; embryonic pattern specification; endoderm development; evasion of host defenses by virus; gastrulation; heart looping; immune response; immune system development; in utero embryonic development; intercellular junction assembly and maintenance; liver development; mesoderm formation; negative regulation of apoptosis; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of fat cell differentiation; negative regulation of inflammatory response; negative regulation of mitotic cell cycle; negative regulation of osteoblast differentiation; negative regulation of osteoblast proliferation; negative regulation of protein catabolic process; negative regulation of transcription from RNA polymerase II promoter; osteoblast development; osteoblast differentiation; paraxial mesoderm morphogenesis; pericardium development; positive regulation of bone mineralization; positive regulation of cell migration; positive regulation of chondrocyte differentiation; positive regulation of focal adhesion formation; positive regulation of interleukin-1 beta production; positive regulation of nitric oxide biosynthetic process; positive regulation of positive chemotaxis; positive regulation of stress fiber formation; positive regulation of transcription factor import into nucleus; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of transforming growth factor-beta3 production; protein stabilization; regulation of binding; regulation of epithelial cell proliferation; regulation of immune response; regulation of striated muscle development; regulation of transcription from RNA polymerase II promoter; regulation of transforming growth factor beta receptor signaling pathway; regulation of transforming growth factor-beta2 production; response to hypoxia; skeletal development; SMAD protein complex assembly; somitogenesis; T cell activation; thyroid gland development; transforming growth factor beta receptor signaling pathway; transport; ureteric bud development; Wnt receptor signaling pathway through beta-catenin |
| NCBI Summary: | transcription factor; important component of the TGF-beta signaling pathway [RGD, Feb 2006] |
| UniProt Code: | P84025 |
| NCBI GenInfo Identifier: | 6981174 |
| NCBI Gene ID: | 25631 |
| NCBI Accession: | NP_037227.1 |
| UniProt Secondary Accession: | P84025,O09064, O09144, O14510, O35273, Q92940, Q93002 Q9GKR4, |
| UniProt Related Accession: | P84025 |
| Molecular Weight: | 48,081 Da |
| NCBI Full Name: | mothers against decapentaplegic homolog 3 |
| NCBI Synonym Full Names: | SMAD family member 3 |
| NCBI Official Symbol: | Smad3 |
| NCBI Official Synonym Symbols: | mad3; Madh3; Smad 3 |
| NCBI Protein Information: | mothers against decapentaplegic homolog 3 |
| UniProt Protein Name: | Mothers against decapentaplegic homolog 3 |
| UniProt Synonym Protein Names: | SMAD family member 3; SMAD 3; Smad3 |
| Protein Family: | Mothers against decapentaplegic |
| UniProt Gene Name: | Smad3 |
| 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. |
