Rat Signaling ELISA Kits 3
Rat GATA4 (GATA Binding Protein 4) CLIA Kit (RTES00234)
- SKU:
- RTES00234
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 37.5pg/mL
- Range:
- 62.5-4000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
- 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: | Rat |
| 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 Rat GATA4 in samples. No significant cross-reactivity or interference between Rat GATA4 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 Rat GATA4. 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 Rat GATA4 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 Rat GATA4, 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 Rat GATA4. The concentration of Rat GATA4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | GATA4: a conserved and ubiquitous member of the GATA family of zinc-finger transcription factors. Members of this family recognize the GATA motif which is present in the promoters of many genes. This protein is thought to regulate genes involved in embryogenesis and in myocardial differentiation and function. May regulate a set of cardiac-specific genes and play a crucial role in cardiogenesis. Defects are a cause of atrial septal defect 2 (ASD2). ASD2 is an autosomal dominant condition with atrial septal defect and other congenital heart disease but no conduction defects or noncardiac abnormalities. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Transcription factor Chromosomal Location of Human Ortholog: 15p12 Cellular Component: nuclear body; nuclear chromatin; nucleus; RNA polymerase II transcription factor complex; transcription factor complex Molecular Function:activating transcription factor binding; chromatin binding; co-SMAD binding; DNA binding; DNA binding transcription factor activity; NFAT protein binding; protein kinase binding; sequence-specific DNA binding; transcription coactivator activity; transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding; transcription factor binding; transcription regulatory region DNA binding; zinc ion binding Biological Process: atrial septum primum morphogenesis; atrial septum secundum morphogenesis; atrioventricular valve formation; atrioventricular valve morphogenesis; BMP signaling pathway; cardiac muscle cell differentiation; cardiac muscle hypertrophy in response to stress; cardiac muscle tissue development; cardiac right ventricle morphogenesis; cardiac septum development; cell-cell signaling; cellular response to follicle-stimulating hormone stimulus; cellular response to glucose stimulus; cellular response to gonadotropin stimulus; embryonic digestive tract morphogenesis; embryonic foregut morphogenesis; embryonic heart tube anterior/posterior pattern formation; embryonic heart tube development; embryonic morphogenesis; endocardial cushion development; endoderm formation; epithelial cell fate commitment; gastrulation with mouth forming second; heart development; heart looping; heart morphogenesis; in utero embryonic development; male gonad development; negative regulation of apoptosis; negative regulation of apoptotic signaling pathway; negative regulation of autophagy; negative regulation of cardiac muscle cell apoptotic process; negative regulation of connective tissue replacement; negative regulation of gene expression; positive regulation of angiogenesis; positive regulation of BMP signaling pathway; positive regulation of cardiac muscle cell proliferation; positive regulation of cardioblast differentiation; positive regulation of cell cycle; positive regulation of ERK1 and ERK2 cascade; positive regulation of gene expression; positive regulation of protein phosphorylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-templated; positive regulation of vascular endothelial growth factor production; regulation of cardiac muscle cell contraction; regulation of cardiac muscle cell proliferation; regulation of gene expression; regulation of protein kinase B signaling; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-templated; response to drug; response to estrogen; response to mechanical stimulus; response to retinoic acid; response to vitamin A; seminiferous tubule development; Sertoli cell differentiation; spermatogenesis; transcription from RNA polymerase II promoter; tube morphogenesis; ventricular cardiac muscle tissue development; ventricular septum development; Wnt receptor signaling pathway through beta-catenin |
| NCBI Summary: | plays a role in regulation of cardiac-myocyte specific gene expression; interacts with dHAND (HAND2) to activate transcription of several cardiac-specific genes [RGD, Feb 2006] |
| UniProt Code: | P46152 |
| NCBI GenInfo Identifier: | 25282465 |
| NCBI Gene ID: | 54254 |
| NCBI Accession: | NP_653331. 1 |
| UniProt Related Accession: | P46152 |
| Molecular Weight: | 44,597 Da |
| NCBI Full Name: | transcription factor GATA-4 |
| NCBI Synonym Full Names: | GATA binding protein 4 |
| NCBI Official Symbol: | Gata4 |
| NCBI Protein Information: | transcription factor GATA-4 |
| UniProt Protein Name: | Transcription factor GATA-4 |
| UniProt Synonym Protein Names: | DNA-binding protein GATA-GT2; GATA-binding factor 4 |
| Protein Family: | GATA transcription factor |
| UniProt Gene Name: | Gata4 |
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 | 58304 59858 | 59081 | 59046 |
| 2000 | 27129 28589 | 27859 | 27824 |
| 1000 | 13741 13227 | 13484 | 13449 |
| 500 | 6031 7179 | 6605 | 6570 |
| 250 | 3331 3155 | 3243 | 3208 |
| 125 | 1650 1512 | 1581 | 1546 |
| 62.50 | 741 769 | 755 | 720 |
| 0 | 34 36 | 35 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat GATA4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat GATA4 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) | 208.75 | 555.99 | 1341.72 | 192.01 | 561.83 | 1305.56 |
| Standard deviation | 22.06 | 56.32 | 150.54 | 18.74 | 42.92 | 116.06 |
| C V (%) | 10.57 | 10.13 | 11.22 | 9.76 | 7.64 | 8.89 |
Recovery
The recovery of Rat GATA4 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) | 85-96 | 90 |
| EDTA plasma (n=5) | 99-115 | 107 |
| Cell culture media (n=5) | 101-115 | 107 |
Linearity
Samples were spiked with high concentrations of Rat GATA4 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 (%) | 99-116 | 101-117 | 84-99 |
| Average (%) | 107 | 109 | 90 | |
| 1:4 | Range (%) | 94-107 | 98-115 | 95-110 |
| Average (%) | 100 | 105 | 100 | |
| 1:8 | Range (%) | 86-98 | 86-96 | 84-98 |
| Average (%) | 93 | 91 | 91 | |
| 1:16 | Range (%) | 97-111 | 88-104 | 86-99 |
| Average (%) | 103 | 96 | 94 |
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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