Mouse Sonic Hedgehog Homolog/SHH ELISA Kit- High Sensitivity

Product Name:	Mouse Sonic Hedgehog Homolog / SHH ELISA Kit
Product Code:	MOFI01109
Size:	96 Assays
Alias:	SHH, HHG1, HHG-1, HLP3, HPE3, SMMCIsonic hedgehog homolog, Drosophila, sonic hedgehog, sonic hedgehog homolog, sonic hedgehog protein, TPT, TPTPS
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse SHH concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse SHH and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse SHH in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	93
EDTA plasma(n=5)	88-105	96
UFH plasma(n=5)	87-105	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse SHH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	88-100%	85-105%	85-105%
EDTA plasma(n=5)	86-100%	82-101%	83-97%
UFH plasma(n=5)	90-100%	81-97%	81-90%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q62226

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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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Mouse Sonic Hedgehog Homolog / SHH ELISA Kit

Description