Mouse Cell Signalling ELISA Kits 2
Mouse SHH (Hedgehog Homolog, Sonic) CLIA Kit (MOES00331)
- SKU:
- MOES00331
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 4.69pg/mL
- Range:
- 7.81-500pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- HHG1, HLP3, HPE3, MCOPCB5, SMMCI, TPT, TPTPS, Sonic hedgehog
- Reactivity:
- Mouse
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| 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 Mouse SHH in samples. No significant cross-reactivity or interference between Mouse SHH 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 SHH. 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 SHH 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 SHH, 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 SHH. The concentration of Mouse SHH in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | SHH: Binds to the patched (PTC) receptor, which functions in association with smoothened (SMO), to activate the transcription of target genes. In the absence of SHH, PTC represses the constitutive signaling activity of SMO. Also regulates another target, the gli oncogene. Intercellular signal essential for a variety of patterning events during development: signal produced by the notochord that induces ventral cell fate in the neural tube and somites, and the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. Displays both floor plate- and motor neuron-inducing activity. The threshold concentration of N-product required for motor neuron induction is 5-fold lower than that required for floor plate induction. Interacts with HHATL/GUP1 which negatively regulates HHAT-mediated palmitoylation of the SHH N-terminus. N-product is active as a multimer. Expressed in fetal intestine, liver, lung, and kidney. Not expressed in adult tissues. Belongs to the hedgehog family. |
| UniProt Protein Details: | Protein type:Oncoprotein; Cell development/differentiation; Cell cycle regulation; Motility/polarity/chemotaxis Cellular Component: axon; cell soma; cell surface; dendrite; endoplasmic reticulum; extracellular space; Golgi apparatus; lipid raft; membrane; proteinaceous extracellular matrix; transport vesicle Molecular Function:calcium ion binding; glycoprotein binding; glycosaminoglycan binding; hydrolase activity; laminin-1 binding; patched binding; protein binding; zinc ion binding Biological Process: activation of hh target transcription factor; anatomical structure development; anatomical structure formation; androgen metabolic process; angiogenesis; anterior/posterior pattern formation; axon guidance; Bergmann glial cell differentiation; blood coagulation; branching morphogenesis of a tube; camera-type eye development; CD4-positive or CD8-positive, alpha-beta T cell lineage commitment; cell development; cell fate commitment; cell fate specification; cell proliferation; cell proliferation in the external granule layer; cell-cell signaling; central nervous system development; determination of left/right symmetry; developmental growth; digestive tract morphogenesis; dorsal/ventral pattern formation; dorsoventral neural tube patterning; ectoderm development; embryonic development; embryonic digestive tract morphogenesis; embryonic digit morphogenesis; embryonic foregut morphogenesis; embryonic forelimb morphogenesis; embryonic hindlimb morphogenesis; embryonic limb morphogenesis; embryonic morphogenesis; embryonic organ development; embryonic skeletal development; endocytosis; establishment of cell polarity; forebrain development; forebrain regionalization; formation of anatomical boundary; granule cell precursor proliferation; gut mesoderm development; hair follicle development; hair follicle morphogenesis; heart development; heart looping; hindbrain development; hindgut morphogenesis; inner ear development; intermediate filament organization; kidney development; limb bud formation; limb development; lung development; lymphoid progenitor cell differentiation; male genitalia development; male genitalia morphogenesis; mesenchymal cell proliferation; metanephros development; midbrain development; myoblast differentiation; myotube differentiation; negative regulation of alpha-beta T cell differentiation; negative regulation of apoptosis; negative regulation of cell differentiation; negative regulation of cell migration; negative regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of protein catabolic process; negative regulation of T cell proliferation; negative regulation of transcription from RNA polymerase II promoter; negative regulation of Wnt receptor signaling pathway; negative thymic T cell selection; neural crest cell migration; neural tube formation; neuroblast proliferation; neuron fate commitment; odontogenesis; odontogenesis of dentine-containing teeth; oligodendrocyte development; oligodendrocyte differentiation; organ formation; osteoblast development; palate development; pancreas development; pattern specification process; patterning of blood vessels; polarity specification of anterior/posterior axis; positive regulation of alpha-beta T cell differentiation; positive regulation of cell differentiation; positive regulation of cell division; positive regulation of cell proliferation; positive regulation of granule cell precursor proliferation; positive regulation of immature T cell proliferation in the thymus; positive regulation of mesenchymal cell proliferation; positive regulation of neuroblast proliferation; positive regulation of neuron differentiation; positive regulation of oligodendrocyte differentiation; positive regulation of photoreceptor cell differentiation; positive regulation of protein import into nucleus; positive regulation of skeletal muscle cell proliferation; positive regulation of skeletal muscle development; positive regulation of smoothened signaling pathway; positive regulation of striated muscle cell differentiation; positive regulation of T cell differentiation in the thymus; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of Wnt receptor signaling pathway; positive thymic T cell selection; prostate gland development; regulation of cell proliferation; regulation of gene expression; regulation of odontogenesis; regulation of proteolysis; regulation of transcription, DNA-dependent; respiratory tube development; response to axon injury; response to ethanol; signal transduction; skin development; smoothened signaling pathway; smoothened signaling pathway in regulation of granule cell precursor cell proliferation; spinal cord dorsal/ventral patterning; spinal cord motor neuron differentiation; stem cell development; striated muscle cell differentiation; striated muscle development; T cell differentiation in the thymus; telencephalon regionalization; thalamus development; thymus development; thyroid gland development; tongue development; tongue morphogenesis; ureteric bud branching; vasculature development; vasculogenesis; ventral spinal cord interneuron specification; Wnt receptor signaling pathway through beta-catenin |
| UniProt Code: | Q62226 |
| NCBI GenInfo Identifier: | 6094284 |
| NCBI Gene ID: | 20423 |
| NCBI Accession: | Q62226. 2 |
| UniProt Related Accession: | Q62226 |
| Molecular Weight: | 47,773 Da |
| NCBI Full Name: | Sonic hedgehog protein |
| NCBI Synonym Full Names: | sonic hedgehog |
| NCBI Official Symbol: | Shh |
| NCBI Official Synonym Symbols: | Hx; Dsh; Hhg1; Hxl3; M100081; 9530036O11Rik |
| NCBI Protein Information: | sonic hedgehog protein |
| UniProt Protein Name: | Sonic hedgehog protein |
| UniProt Synonym Protein Names: | HHG-1 |
| Protein Family: | Sonic hedgehog protein |
| UniProt Gene Name: | Shh |
| UniProt Entry Name: | SHH_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 |
| 500 | 50610 52464 | 51537 | 51508 |
| 250 | 20310 21686 | 20998 | 20969 |
| 125 | 10137 8655 | 9396 | 9367 |
| 62.5 | 4340 4684 | 4512 | 4483 |
| 31.25 | 2324 2274 | 2299 | 2270 |
| 15.63 | 1336 1164 | 1250 | 1221 |
| 7.81 | 704 776 | 740 | 711 |
| 0 | 28 30 | 29 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse SHH were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse SHH 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) | 27.19 | 65.91 | 197.50 | 25.90 | 64.46 | 196.25 |
| Standard deviation | 2.18 | 7.71 | 19.65 | 2.89 | 4.64 | 21.76 |
| C V (%) | 8.02 | 11.70 | 9.95 | 11.16 | 7.20 | 11.09 |
Recovery
The recovery of Mouse SHH 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) | 98-114 | 106 |
| EDTA plasma (n=5) | 99-114 | 106 |
| Cell culture media (n=5) | 90-104 | 98 |
Linearity
Samples were spiked with high concentrations of Mouse SHH 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 (%) | 102-116 | 88-100 | 95-106 |
| Average (%) | 109 | 94 | 100 | |
| 1:4 | Range (%) | 88-100 | 92-104 | 90-103 |
| Average (%) | 95 | 98 | 96 | |
| 1:8 | Range (%) | 96-114 | 92-105 | 93-108 |
| Average (%) | 104 | 99 | 100 | |
| 1:16 | Range (%) | 87-100 | 89-101 | 89-99 |
| Average (%) | 94 | 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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