Mouse Cardiovascular ELISA Kits
Mouse VEGFR3/Flt4 (Vascular Endothelial Cell Growth Factor Receptor 3) CLIA Kit (MOES00592)
- SKU:
- MOES00592
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Mouse
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cardiovascular
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection method: | Chemiluminescence |
| Detection range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Mouse VEGFR3/Flt4 in samples. No significant cross-reactivity or interference between Mouse VEGFR3/Flt4 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 Mouse VEGFR3/Flt4. 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 Mouse VEGFR3/Flt4 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 Mouse VEGFR3/Flt4, 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 Mouse VEGFR3/Flt4. The concentration of Mouse VEGFR3/Flt4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | VEGFR3: Tyrosine-protein kinase that acts as a cell-surface receptor for VEGFC and VEGFD, and plays an essential role in adult lymphangiogenesis and in the development of the vascular network and the cardiovascular system during embryonic development. Promotes proliferation, survival and migration of endothelial cells, and regulates angiogenic sprouting. Signaling by activated FLT4 leads to enhanced production of VEGFC, and to a lesser degree VEGFA, thereby creating a positive feedback loop that enhances FLT4 signaling. Modulates KDR signaling by forming heterodimers. The secreted isoform 3 may function as a decoy receptor for VEGFC and/or VEGFD and play an important role as a negative regulator of VEGFC-mediated lymphangiogenesis and angiogenesis. Binding of vascular growth factors to isoform 1 or isoform 2 leads to the activation of several signaling cascades; isoform 2 seems to be less efficient in signal transduction, because it has a truncated C-terminus and therefore lacks several phosphorylation sites. Mediates activation of the MAPK1/ERK2, MAPK3/ERK1 signaling pathway, of MAPK8 and the JUN signaling pathway, and of the AKT1 signaling pathway. Phosphorylates SHC1. Mediates phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3- kinase. Promotes phosphorylation of MAPK8 at 'Thr-183' and 'Tyr- 185', and of AKT1 at 'Ser-473'. Interacts with VEGFC and VEGFD. Monomer in the absence of bound VEGFC or VEGFD. Homodimer in the presence of bound VEGFC or VEGFD. Can also form a heterodimer with KDR. Interacts with PTPN14; the interaction is enhanced by stimulation with VEGFC. Interacts with CRK, GRB2, PTK2/FAK1, SHC1, PIK3R1 and PTPN11/SHP- 2. Identified in a complex with SRC and ITGB1. Detected in endothelial cells. Widely expressed. Detected in fetal spleen, lung and brain. Detected in adult liver, muscle, thymus, placenta, lung, testis, ovary, prostate, heart, and kidney. Present in an inactive conformation in the absence of bound ligand. Binding of VEGFC or VEGFD leads to dimerization and activation by autophosphorylation on tyrosine residues. Inhibited by MAZ51. Belongs to the protein kinase superfamily. Tyr protein kinase family. CSF-1/PDGF receptor subfamily. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Protein kinase, TK; Kinase, protein; Membrane protein, integral; EC 2. 7. 10. 1; Protein kinase, tyrosine (receptor); TK group; VEGFR family Cellular Component: cytoplasm; integral to membrane; integral to plasma membrane; membrane; nucleus; plasma membrane; receptor complex Molecular Function:ATP binding; growth factor binding; kinase activity; nucleotide binding; protein binding; protein kinase activity; protein phosphatase binding; protein-tyrosine kinase activity; transferase activity; transmembrane receptor protein tyrosine kinase activity; vascular endothelial growth factor receptor activity Biological Process: alveolus development; angiogenesis; blood vessel morphogenesis; lymph vessel development; lymphangiogenesis; negative regulation of apoptosis; peptidyl-tyrosine phosphorylation; phosphorylation; positive regulation of cell growth; positive regulation of cell proliferation; positive regulation of endothelial cell proliferation; positive regulation of JNK cascade; positive regulation of MAPKKK cascade; positive regulation of protein amino acid phosphorylation; protein amino acid autophosphorylation; protein amino acid phosphorylation; respiratory gaseous exchange; respiratory system process; sprouting angiogenesis; transmembrane receptor protein tyrosine kinase signaling pathway; vascular endothelial growth factor receptor signaling pathway; vasculature development |
| UniProt Code: | P35917 |
| NCBI GenInfo Identifier: | 544322 |
| NCBI Gene ID: | 14257 |
| NCBI Accession: | P35917. 1 |
| UniProt Related Accession: | P35917 |
| Molecular Weight: | 153,016 Da |
| NCBI Full Name: | Vascular endothelial growth factor receptor 3 |
| NCBI Synonym Full Names: | FMS-like tyrosine kinase 4 |
| NCBI Official Symbol: | Flt4 |
| NCBI Official Synonym Symbols: | Chy; Flt-4; VEGFR3; VEGFR-3; AI323512 |
| NCBI Protein Information: | vascular endothelial growth factor receptor 3 |
| UniProt Protein Name: | Vascular endothelial growth factor receptor 3 |
| UniProt Synonym Protein Names: | Fms-like tyrosine kinase 4; FLT-4; Tyrosine-protein kinase receptor FLT4 |
| Protein Family: | Vascular endothelial growth factor receptor |
| UniProt Gene Name: | Flt4 |
| UniProt Entry Name: | VGFR3_MOUSE |
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 |
| 1000 | 51187 56163 | 53675 | 53650 |
| 500 | 21711 23267 | 22489 | 22464 |
| 250 | 10668 9754 | 10211 | 10186 |
| 125 | 4596 5206 | 4901 | 4876 |
| 62.5 | 2483 2423 | 2453 | 2428 |
| 31.25 | 1344 1218 | 1281 | 1256 |
| 15.63 | 661 755 | 708 | 683 |
| 0 | 24 26 | 25 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse VEGFR3/Flt4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse VEGFR3/Flt4 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) | 47.54 | 153.41 | 358.82 | 43.42 | 151.94 | 390.32 |
| Standard deviation | 4.30 | 14.41 | 32.33 | 4.33 | 17.17 | 27.21 |
| C V (%) | 9.05 | 9.39 | 9.01 | 9.97 | 11.30 | 6.97 |
Recovery
The recovery of Mouse VEGFR3/Flt4 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) | 84-95 | 90 |
| EDTA plasma (n=5) | 94-108 | 102 |
| Cell culture media (n=5) | 94-108 | 101 |
Linearity
Samples were spiked with high concentrations of Mouse VEGFR3/Flt4 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 (%) | 96-112 | 90-104 | 98-112 |
| Average (%) | 102 | 97 | 106 | |
| 1:4 | Range (%) | 93-105 | 101-118 | 102-114 |
| Average (%) | 99 | 109 | 108 | |
| 1:8 | Range (%) | 88-100 | 98-115 | 95-107 |
| Average (%) | 95 | 105 | 102 | |
| 1:16 | Range (%) | 85-96 | 98-114 | 93-109 |
| Average (%) | 90 | 104 | 100 |
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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