Mouse Sonic hedgehog protein (Shh) ELISA Kit
- SKU:
- MOEB1720
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q62226
- ELISA Type:
- Sandwich
- Synonyms:
- SHH, HHG1, HHG-1, HLP3, HPE3, SMMCIsonic hedgehog homolog, Drosophila, sonic hedgehog, sonic hedgehog homolog, sonic hedgehog protein, TPT, TPTPS
- Reactivity:
- Mouse
Description
| Product Name: | Mouse Sonic hedgehog protein (Shh) ELISA Kit |
| Product Code: | MOEB1720 |
| Alias: | Sonic hedgehog protein, SHH, HHG-1, Shh, Hhg1 |
| Uniprot: | Q62226 |
| Reactivity: | Mouse |
| Range: | Please contact us for more information |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| 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 |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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