Rat Signaling ELISA Kits 3
Rat SCF (Stem Cell Factor) CLIA Kit (RTES00526)
- SKU:
- RTES00526
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 4.69pg/mL
- Range:
- 7.81-500pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- KITLG, FPH2, KL-1, Kitl, MGF, SF, SHEP7
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection method: | Chemiluminescence |
| Detection range: | 7.81-500 pg/mL |
| Sensitivity: | 4.69 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Rat SCF in samples. No significant cross-reactivity or interference between Rat SCF 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 SCF. 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 SCF 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 SCF, 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 SCF. The concentration of Rat SCF in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | SCF: Ligand for the receptor-type protein-tyrosine kinase KIT. Plays an essential role in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and in melanogenesis. KITLG/SCF binding can activate several signaling pathways. Promotes phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, and subsequent activation of the kinase AKT1. KITLG/SCF and KIT also transmit signals via GRB2 and activation of RAS, RAF1 and the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1. KITLG/SCF and KIT promote activation of STAT family members STAT1, STAT3 and STAT5. KITLG/SCF and KIT promote activation of PLCG1, leading to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5- trisphosphate. KITLG/SCF acts synergistically with other cytokines, probably interleukins. Homodimer, non-covalently linked (Probable). Heterotetramer with KIT, binding two KIT molecules; thereby mediates KIT dimerization and subsequent activation by autophosphorylation. Belongs to the SCF family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral Cellular Component: extracellular space; cytoskeleton; membrane; cytoplasm; extracellular region; integral to membrane; plasma membrane Molecular Function:growth factor activity; cytokine activity; stem cell factor receptor binding Biological Process: positive regulation of MAP kinase activity; positive regulation of peptidyl-tyrosine phosphorylation; ovarian follicle development; positive regulation of melanocyte differentiation; negative regulation of mast cell apoptosis; embryonic hemopoiesis; positive regulation of cell proliferation; germ cell programmed cell death; male gonad development; positive regulation of Ras protein signal transduction; positive regulation of myeloid leukocyte differentiation; cell adhesion; neural crest cell migration; positive regulation of DNA replication; negative regulation of apoptosis |
| NCBI Summary: | soluble growth factor that activates c-kit tyrosine kinase receptor; important for signaling in spermatogenesis; putative regulator of Leydig cell development [RGD, Feb 2006] |
| UniProt Code: | P21581 |
| NCBI GenInfo Identifier: | 22654283 |
| NCBI Gene ID: | 60427 |
| NCBI Accession: | P21581. 2 |
| UniProt Secondary Accession: | P21581,Q9QWZ4, Q9Z2E7, |
| UniProt Related Accession: | P21581 |
| Molecular Weight: | 27,681 Da |
| NCBI Full Name: | Kit ligand |
| NCBI Synonym Full Names: | KIT ligand |
| NCBI Official Symbol: | Kitlg |
| NCBI Official Synonym Symbols: | Mgf; SCF; Kitl |
| NCBI Protein Information: | kit ligand; C-kit ligand; stem cell factor KL-1; mast cell growth factor; steel factor/kit ligand; hematopoietic growth factor KL |
| UniProt Protein Name: | Kit ligand |
| UniProt Synonym Protein Names: | Hematopoietic growth factor KL; Mast cell growth factor; MGF; Stem cell factor; SCF; c-Kit ligandCleaved into the following chain:Soluble KIT ligand; sKITLG |
| Protein Family: | SCF-associated factor |
| UniProt Gene Name: | Kitlg |
| UniProt Entry Name: | SCF_RAT |
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 |
| 500 | 47654 55420 | 51537 | 51508 |
| 250 | 19281 22715 | 20998 | 20969 |
| 125 | 9969 8823 | 9396 | 9367 |
| 62.5 | 4326 4698 | 4512 | 4483 |
| 31.25 | 2475 2123 | 2299 | 2270 |
| 15.63 | 1267 1233 | 1250 | 1221 |
| 7.81 | 733 747 | 740 | 711 |
| 0 | 29 29 | 29 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat SCF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat SCF 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) | 26.61 | 69.24 | 167.83 | 24.72 | 64.80 | 161.14 |
| Standard deviation | 3.43 | 5.03 | 16.25 | 2.70 | 4.70 | 18.31 |
| C V (%) | 12.89 | 7.26 | 9.68 | 10.92 | 7.25 | 11.36 |
Recovery
The recovery of Rat SCF 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) | 98-112 | 104 |
| EDTA plasma (n=5) | 94-106 | 100 |
| Cell culture media (n=5) | 99-113 | 106 |
Linearity
Samples were spiked with high concentrations of Rat SCF 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 (%) | 98-114 | 98-115 | 94-107 |
| Average (%) | 106 | 105 | 102 | |
| 1:4 | Range (%) | 99-115 | 99-117 | 95-109 |
| Average (%) | 105 | 107 | 103 | |
| 1:8 | Range (%) | 96-112 | 84-98 | 101-111 |
| Average (%) | 102 | 91 | 106 | |
| 1:16 | Range (%) | 92-104 | 98-114 | 87-99 |
| Average (%) | 97 | 106 | 93 |
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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