Product Name:	Human Sonic hedgehog protein / SHH ELISA Kit
Product Code:	HUFI00439
Size:	96 Assays
Alias:	SHH, Shh, HHG1, HHG-1, HLP3, HPE3, SMMCIsonic hedgehog homolog, Drosophila, sonic hedgehog, sonic hedgehog homolog, sonic hedgehog protein, TPT, TPTPS
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human SHH concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-105	96
EDTA plasma(n=5)	86-96	93
UFH plasma(n=5)	91-97	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human 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)	89-100%	93-105%	91-103%
EDTA plasma(n=5)	86-96%	82-99%	82-100%
UFH plasma(n=5)	93-98%	83-100%	86-99%

CV(%):

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

Q15465

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:Motility/polarity/chemotaxis; Cell cycle regulation; Oncoprotein; Cell development/differentiation Chromosomal Location of Human Ortholog: 7q36 Cellular Component: extracellular matrix; extracellular space; cell surface; endoplasmic reticulum lumen; plasma membrane; extracellular region; nucleus; cytosol; lipid raft Molecular Function:laminin-1 binding; peptidase activity; morphogen activity; protein binding; glycosaminoglycan binding; zinc ion binding; patched binding; calcium ion binding; glycoprotein binding Biological Process: prostate gland development; central nervous system development; positive regulation of transcription, DNA-dependent; embryonic skeletal development; telencephalon regionalization; embryonic foregut morphogenesis; male genitalia development; neural crest cell migration; inner ear development; embryonic limb morphogenesis; hindbrain development; positive regulation of neuroblast proliferation; camera-type eye development; neuron fate commitment; myotube differentiation; intermediate filament organization; osteoblast development; positive regulation of skeletal muscle cell proliferation; positive regulation of cell division; regulation of proteolysis; positive regulation of transcription from RNA polymerase II promoter; embryonic digit morphogenesis; negative regulation of apoptosis; embryonic forelimb morphogenesis; axon guidance; granule cell precursor proliferation; ventral midline development; spinal cord dorsal/ventral patterning; thalamus development; positive regulation of striated muscle cell differentiation; negative regulation of transcription from RNA polymerase II promoter; palate development; Bergmann glial cell differentiation; negative regulation of T cell proliferation; positive regulation of cell proliferation; pancreas development; forebrain development; thyroid gland development; heart looping; vasculogenesis; negative regulation of cell migration; positive thymic T cell selection; intein-mediated protein splicing; regulation of odontogenesis; androgen metabolic process; spinal cord motor neuron differentiation; pattern specification process; regulation of cell proliferation; odontogenesis of dentine-containing teeth; negative regulation of cell differentiation; stem cell development; embryonic development; dorsal/ventral pattern formation; positive regulation of protein import into nucleus; ureteric bud branching; hindgut morphogenesis; lung development; negative regulation of alpha-beta T cell differentiation; heart development; T cell differentiation in the thymus; CD4-positive or CD8-positive, alpha-beta T cell lineage commitment; lymphoid progenitor cell differentiation; Wnt receptor signaling pathway through beta-catenin; embryonic pattern specification; proteolysis; positive regulation of T cell differentiation in the thymus; cell-cell signaling; embryonic digestive tract morphogenesis; midbrain development; ectoderm development; positive regulation of smoothened signaling pathway; positive regulation of oligodendrocyte differentiation; oligodendrocyte development; activation of hh target transcription factor; striated muscle development; endocytosis; positive regulation of skeletal muscle development; negative thymic T cell selection; patterning of blood vessels; branching morphogenesis of a tube; polarity specification of anterior/posterior axis; metanephros development; cell fate specification; embryonic hindlimb morphogenesis; positive regulation of Wnt receptor signaling pathway; dorsoventral neural tube patterning; smoothened signaling pathway; organ formation; hair follicle morphogenesis; thymus development; smoothened signaling pathway in regulation of granule cell precursor cell proliferation; positive regulation of immature T cell proliferation in the thymus; negative regulation of proteasomal ubiquitin-dependent protein catabolic process; formation of anatomical boundary; myoblast differentiation; limb bud formation; establishment of cell polarity; neuroblast proliferation; blood coagulation; cell development; positive regulation of alpha-beta T cell differentiation Disease: Solitary Median Maxillary Central Incisor; Schizencephaly; Microphthalmia, Isolated, With Coloboma 5; Holoprosencephaly 3
NCBI Summary:	This gene encodes a protein that is instrumental in patterning the early embryo. It has been implicated as the key inductive signal in patterning of the ventral neural tube, the anterior-posterior limb axis, and the ventral somites. Of three human proteins showing sequence and functional similarity to the sonic hedgehog protein of Drosophila, this protein is the most similar. The protein is made as a precursor that is autocatalytically cleaved; the N-terminal portion is soluble and contains the signalling activity while the C-terminal portion is involved in precursor processing. More importantly, the C-terminal product covalently attaches a cholesterol moiety to the N-terminal product, restricting the N-terminal product to the cell surface and preventing it from freely diffusing throughout the developing embryo. Defects in this protein or in its signalling pathway are a cause of holoprosencephaly (HPE), a disorder in which the developing forebrain fails to correctly separate into right and left hemispheres. HPE is manifested by facial deformities. It is also thought that mutations in this gene or in its signalling pathway may be responsible for VACTERL syndrome, which is characterized by vertebral defects, anal atresia, tracheoesophageal fistula with esophageal atresia, radial and renal dysplasia, cardiac anomalies, and limb abnormalities. Additionally, mutations in a long range enhancer located approximately 1 megabase upstream of this gene disrupt limb patterning and can result in preaxial polydactyly. [provided by RefSeq, Jul 2008]
UniProt Code:	Q15465
NCBI GenInfo Identifier:	6094283
NCBI Gene ID:	6469
NCBI Accession:	Q15465.1
UniProt Secondary Accession:	Q15465,Q75MC9, A4D247,
UniProt Related Accession:	Q15465
Molecular Weight:	49,607 Da
NCBI Full Name:	Sonic hedgehog protein
NCBI Synonym Full Names:	sonic hedgehog
NCBI Official Symbol:	SHHÃ‚ Ã‚
NCBI Official Synonym Symbols:	TPT; HHG1; HLP3; HPE3; SMMCI; TPTPS; MCOPCB5Ã‚ Ã‚
NCBI Protein Information:	sonic hedgehog protein; sonic hedgehog homolog
UniProt Protein Name:	Sonic hedgehog protein
UniProt Synonym Protein Names:	HHG-1Cleaved into the following 2 chains:Sonic hedgehog protein N-product; Sonic hedgehog protein C-product
Protein Family:	Sonic hedgehog protein
UniProt Gene Name:	SHHÃ‚ Ã‚
UniProt Entry Name:	SHH_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 Ã‚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 Ã‚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.