Human Smad4/MADH4 (Mothers Against Decapentaplegic Homolog 4 )CLIA Kit (HUES00556)
- SKU:
- HUES00556
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 37.5pg/mL
- Range:
- 62.5-4000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Epigenetics and Nuclear Signaling
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human Smad4/MADH4 in samples. No significant cross-reactivity or interference between Human Smad4/MADH4 and analogues was observed. |
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human Smad4/MADH4. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human Smad4/MADH4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human Smad4/MADH4, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human Smad4/MADH4. The concentration of Human Smad4/MADH4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | SMAD4: transcription factor that mediates signal transduction by the transforming growth factor superfamily. The common smad (co-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. Promotes binding of the Smad2/Smad4/Fast-1 complex to DNA and provides an activation function required for Smad1 or Smad2 to stimulate transcription. |
| UniProt Protein Details: | Protein type:Nuclear receptor co-regulator; DNA-binding; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 18q21. 1 Cellular Component: nucleoplasm; centrosome; transcription factor complex; nuclear chromatin; cytoplasm; cytosol; nucleus Molecular Function:collagen binding; identical protein binding; protein binding; transforming growth factor beta receptor, common-partner cytoplasmic mediator activity; protein homodimerization activity; DNA binding; sequence-specific DNA binding; metal ion binding; chromatin binding; transcription factor activity Biological Process: developmental growth; axon guidance; somatic stem cell maintenance; positive regulation of transcription, DNA-dependent; sebaceous gland development; negative regulation of transcription from RNA polymerase II promoter; palate development; BMP signaling pathway; negative regulation of cell proliferation; regulation of transforming growth factor beta receptor signaling pathway; transforming growth factor beta receptor signaling pathway; mesoderm development; neural crest cell differentiation; positive regulation of BMP signaling pathway; transcription initiation from RNA polymerase II promoter; regulation of transforming growth factor-beta2 production; regulation of binding; transcription, DNA-dependent; in utero embryonic development; neuron fate commitment; positive regulation of transforming growth factor beta receptor signaling pathway; gastrulation with mouth forming second; somite rostral/caudal axis specification; SMAD protein complex assembly; formation of anatomical boundary; endothelial cell activation; cell proliferation; ureteric bud branching; response to hypoxia; gene expression; positive regulation of transcription from RNA polymerase II promoter; negative regulation of cell growth; regulation of hair follicle development; negative regulation of protein catabolic process; negative regulation of transcription, DNA-dependent; endoderm development Disease: Pancreatic Cancer; Juvenile Polyposis Syndrome; Myhre Syndrome; Juvenile Polyposis/hereditary Hemorrhagic Telangiectasia Syndrome |
| NCBI Summary: | This gene encodes a member of the Smad family of signal transduction proteins. Smad proteins are phosphorylated and activated by transmembrane serine-threonine receptor kinases in response to TGF-beta signaling. The product of this gene forms homomeric complexes and heteromeric complexes with other activated Smad proteins, which then accumulate in the nucleus and regulate the transcription of target genes. This protein binds to DNA and recognizes an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE). The Smad proteins are subject to complex regulation by post-translational modifications. Mutations or deletions in this gene have been shown to result in pancreatic cancer, juvenile polyposis syndrome, and hereditary hemorrhagic telangiectasia syndrome. [provided by RefSeq, Oct 2009] |
| UniProt Code: | Q13485 |
| NCBI GenInfo Identifier: | 13959561 |
| NCBI Gene ID: | 4089 |
| NCBI Accession: | Q13485. 1 |
| UniProt Secondary Accession: | Q13485,A8K405, |
| UniProt Related Accession: | Q13485 |
| Molecular Weight: | |
| NCBI Full Name: | Mothers against decapentaplegic homolog 4 |
| NCBI Synonym Full Names: | SMAD family member 4 |
| NCBI Official Symbol: | SMAD4 |
| NCBI Official Synonym Symbols: | JIP; DPC4; MADH4; MYHRS |
| NCBI Protein Information: | mothers against decapentaplegic homolog 4; MAD homolog 4; SMAD, mothers against DPP homolog 4; deleted in pancreatic carcinoma locus 4; deletion target in pancreatic carcinoma 4; mothers against decapentaplegic, Drosophila, homolog of, 4 |
| UniProt Protein Name: | Mothers against decapentaplegic homolog 4 |
| UniProt Synonym Protein Names: | Deletion target in pancreatic carcinoma 4; SMAD family member 4; SMAD 4; Smad4; hSMAD4 |
| Protein Family: | Mothers against decapentaplegic |
| UniProt Gene Name: | SMAD4 |
| UniProt Entry Name: | SMAD4_HUMAN |
As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (pg/mL) | RLU | Average | Corrected |
| 4000 | 55621 62541 | 59081 | 59046 |
| 2000 | 27105 28613 | 27859 | 27824 |
| 1000 | 13636 13332 | 13484 | 13449 |
| 500 | 6360 6850 | 6605 | 6570 |
| 250 | 3426 3060 | 3243 | 3208 |
| 125 | 1582 1580 | 1581 | 1546 |
| 62.50 | 732 778 | 755 | 720 |
| 0 | 35 35 | 35 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Smad4/MADH4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Smad4/MADH4 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 188.32 | 465.34 | 1687.45 | 200.65 | 420.11 | 1531.59 |
| Standard deviation | 23.24 | 52.58 | 189.84 | 17.24 | 38.40 | 103.23 |
| C V (%) | 12.34 | 11.30 | 11.25 | 8.59 | 9.14 | 6.74 |
Recovery
The recovery of Human Smad4/MADH4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 96-113 | 104 |
| EDTA plasma (n=5) | 96-108 | 103 |
| Cell culture media (n=5) | 86-100 | 91 |
Linearity
Samples were spiked with high concentrations of Human Smad4/MADH4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 96-111 | 87-103 | 101-114 |
| Average (%) | 102 | 94 | 107 | |
| 1:4 | Range (%) | 91-105 | 95-112 | 89-104 |
| Average (%) | 99 | 103 | 95 | |
| 1:8 | Range (%) | 94-111 | 98-109 | 89-101 |
| Average (%) | 101 | 103 | 94 | |
| 1:16 | Range (%) | 84-97 | 99-116 | 84-96 |
| Average (%) | 91 | 107 | 91 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro CLIA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
- Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- Determine the RLU value of each well immediately.
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