Rat Signaling ELISA Kits 1
Rat Mothers against decapentaplegic homolog 3 (Smad3) ELISA Kit
- SKU:
- RTEB1418
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P84025
- ELISA Type:
- Sandwich
- Synonyms:
- Smad3, MADH3, SMAD family member 3
- Reactivity:
- Rat
Description
| Product Name: | Rat Mothers against decapentaplegic homolog 3 (Smad3) ELISA Kit |
| Product Code: | RTEB1418 |
| Alias: | Mothers against decapentaplegic homolog 3, MAD homolog 3, Mad3, Mothers against DPP homolog 3, SMAD family member 3, SMAD 3, Smad3, Smad3, Madh3 |
| Uniprot: | P84025 |
| Reactivity: | Rat |
| Range: | Please contact us for more information |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| 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. |
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