Product Name:	Human FGF8 ELISA Kit
Product Code:	HUFI01786
Size:	96 Assays
Alias:	FGF8, Androgen-induced growth factor, AIGF, Heparin-binding growth factor 8, HBGF-8, AIGF, HBGF-8, AIGFKAL6, Androgen-induced growth factor, FGF-8, fibroblast growth factor 8
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human FGF8 concentrations in serum plasma and other biological fluids.
Sensitivity:	7.5pg/ml
Range:	12.5-800pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human FGF8 and the recovery rates were calculated by comparing the measured value to the expected amount of Human FGF8 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-105	99
EDTA plasma(n=5)	86-103	94
UFH plasma(n=5)	85-103	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human FGF8 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)	85-105%	87-98%	86-101%
EDTA plasma(n=5)	83-98%	84-99%	84-101%
UFH plasma(n=5)	82-97%	81-99%	81-99%

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

P55075

UniProt Protein Function:	FGF8: Plays an important role in the regulation of embryonic development, cell proliferation, cell differentiation and cell migration. Required for normal brain, eye, ear and limb development during embryogenesis. Required for normal development of the gonadotropin-releasing hormone (GnRH) neuronal system. Defects in FGF8 are the cause of Kallmann syndrome type 6 (KAL6). Kallmann syndrome is a disorder that associates hypogonadotropic hypogonadism and anosmia. Anosmia or hyposmia is related to the absence or hypoplasia of the olfactory bulbs and tracts. Hypogonadism is due to deficiency in gonadotropin-releasing hormone and probably results from a failure of embryonic migration of gonadotropin-releasing hormone- synthesizing neurons. In some patients other developmental anomalies can be present, which include renal agenesis, cleft lip and/or palate, selective tooth agenesis, and bimanual synkinesis. In some cases anosmia may be absent or inconspicuous. Defects in FGF8 are a cause of idiopathic hypogonadotropic hypogonadism (IHH). IHH is defined as a deficiency of the pituitary secretion of follicle-stimulating hormone and luteinizing hormone, which results in the impairment of pubertal maturation and of reproductive function. Belongs to the heparin-binding growth factors family. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted; Cytokine; Secreted, signal peptide Chromosomal Location of Human Ortholog: 10q24 Cellular Component: extracellular space; extracellular region; external side of plasma membrane Molecular Function:growth factor activity; type 2 fibroblast growth factor receptor binding; type 1 fibroblast growth factor receptor binding; fibroblast growth factor receptor binding; chemoattractant activity Biological Process: gonad development; nerve growth factor receptor signaling pathway; apoptosis; cell proliferation in forebrain; adrenocorticotropin hormone secreting cell differentiation; thyroid stimulating hormone secreting cell differentiation; gastrulation; forebrain dorsal/ventral pattern formation; motor axon guidance; Wnt receptor signaling pathway through beta-catenin; mesodermal cell migration; response to organic cyclic substance; embryonic hindlimb morphogenesis; odontogenesis; BMP signaling pathway; positive chemotaxis; induction of an organ; positive regulation of cell proliferation; thyroid gland development; male genitalia development; mesonephros development; pallium development; heart looping; negative regulation of neuron apoptosis; otic vesicle formation; regulation of odontogenesis of dentine-containing teeth; negative regulation of cardiac muscle development; epidermal growth factor receptor signaling pathway; response to drug; anatomical structure morphogenesis; pharyngeal system development; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; positive regulation of mitosis; neural plate morphogenesis; MAPKKK cascade; subpallium development; forebrain morphogenesis; dorsal/ventral axon guidance; positive regulation of organ growth; patterning of blood vessels; ureteric bud branching; forebrain neuron development; midbrain-hindbrain boundary development; insulin receptor signaling pathway; innate immune response; blood vessel remodeling; response to oxidative stress; metanephros development Disease: Hypogonadotropic Hypogonadism 6 With Or Without Anosmia
NCBI Summary:	The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. This protein is known to be a factor that supports androgen and anchorage independent growth of mammary tumor cells. Overexpression of this gene has been shown to increase tumor growth and angiogensis. The adult expression of this gene is restricted to testes and ovaries. Temporal and spatial pattern of this gene expression suggests its function as an embryonic epithelial factor. Studies of the mouse and chick homologs revealed roles in midbrain and limb development, organogenesis, embryo gastrulation and left-right axis determination. The alternative splicing of this gene results in four transcript variants. [provided by RefSeq, Jul 2008]
UniProt Code:	P55075
NCBI GenInfo Identifier:	1706791
NCBI Gene ID:	2253
NCBI Accession:	P55075.1
UniProt Secondary Accession:	P55075,Q14915, Q15766, A1A514,
UniProt Related Accession:	P55075
Molecular Weight:	244
NCBI Full Name:	Fibroblast growth factor 8
NCBI Synonym Full Names:	fibroblast growth factor 8 (androgen-induced)
NCBI Official Symbol:	FGF8
NCBI Official Synonym Symbols:	HH6; AIGF; KAL6; FGF-8; HBGF-8
NCBI Protein Information:	fibroblast growth factor 8; androgen-induced growth factor; heparin-binding growth factor 8
UniProt Protein Name:	Fibroblast growth factor 8
UniProt Synonym Protein Names:	Androgen-induced growth factor; AIGF; Heparin-binding growth factor 8; HBGF-8
UniProt Gene Name:	FGF8
UniProt Entry Name:	FGF8_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 Âµl 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 Âµl 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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Human FGF8 ELISA Kit

Description