Human Smad3/MADH3 (Mothers Against Decapentaplegic Homolog 3) CLIA Kit (HUES00767)
- SKU:
- HUES00767
- Product Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 0.09ng/mL
- Range:
- 0.16-10ng/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Tested Sample Types:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 0.16-10 ng/mL |
| Sensitivity: | 0.09 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human Smad3/MADH3 in samples. No significant cross-reactivity or interference between Human Smad3/MADH3 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 Smad3/MADH3. 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 Smad3/MADH3 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 Smad3/MADH3, 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 Smad3/MADH3. The concentration of Human Smad3/MADH3 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein 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. |
| 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 functions as a transcriptional modulator activated by transforming growth factor-beta and is thought to play a role in the regulation of carcinogenesis. [provided by RefSeq, Apr 2009] |
| UniProt Code: | P84022 |
| NCBI GenInfo Identifier: | 51338669 |
| NCBI Gene ID: | 4088 |
| NCBI Accession: | P84022. 1 |
| UniProt Secondary Accession: | P84022,O09064, O09144, O14510, O35273, Q92940, Q93002 A8K4B6, B7Z4Z5, B7Z6M9, B7Z9Q2, F5H383, |
| UniProt Related Accession: | P84022 |
| Molecular Weight: | 25,722 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: | LDS3; LDS1C; MADH3; JV15-2; HSPC193; HsT17436 |
| NCBI Protein Information: | mothers against decapentaplegic homolog 3 |
| UniProt Protein Name: | Mothers against decapentaplegic homolog 3 |
| UniProt Synonym Protein Names: | JV15-2; SMAD family member 3; SMAD 3; Smad3; hSMAD3 |
| Protein Family: | Mothers against decapentaplegic |
| UniProt Gene Name: | SMAD3 |
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 (ng/mL) | RLU | Average | Corrected |
| 10 | 31788 33154 | 32471 | 32447 |
| 5 | 14896 16044 | 15470 | 15446 |
| 2.5 | 8004 7002 | 7503 | 7479 |
| 1.25 | 3573 3733 | 3653 | 3629 |
| 0.63 | 1805 1717 | 1761 | 1737 |
| 0.31 | 888 760 | 824 | 800 |
| 0.16 | 348 366 | 357 | 333 |
| 0 | 23 25 | 24 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Smad3/MADH3 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Smad3/MADH3 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 (ng/mL) | 0.54 | 1.01 | 3.87 | 0.56 | 1.06 | 4.07 |
| Standard deviation | 0.06 | 0.11 | 0.38 | 0.06 | 0.10 | 0.42 |
| C V (%) | 11.11 | 10.89 | 9.82 | 10.71 | 9.43 | 10.32 |
Recovery
The recovery of Human Smad3/MADH3 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) | 94-109 | 101 |
| EDTA plasma (n=5) | 101-113 | 107 |
| Cell culture media (n=5) | 87-101 | 94 |
Linearity
Samples were spiked with high concentrations of Human Smad3/MADH3 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 (%) | 86-101 | 101-117 | 89-104 |
| Average (%) | 93 | 109 | 96 | |
| 1:4 | Range (%) | 98-112 | 97-111 | 93-105 |
| Average (%) | 105 | 103 | 99 | |
| 1:8 | Range (%) | 93-108 | 93-108 | 93-103 |
| Average (%) | 100 | 100 | 98 | |
| 1:16 | Range (%) | 94-106 | 85-96 | 84-97 |
| Average (%) | 100 | 91 | 90 |
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.
