Human Fibroblast growth factor 8 (FGF8) ELISA Kit

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SKU:
HUEB2203
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P55075
Range:
15.6-1000 pg/mL
ELISA Type:
Sandwich
Synonyms:
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
Reactivity:
Human
Frequently bought together:

Description

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Product Name:Human Fibroblast growth factor 8 (FGF8) ELISA Kit
Product Code:HUEB2203
Alias:Fibroblast growth factor 8, FGF-8, Androgen-induced growth factor, AIGF, Heparin-binding growth factor 8, HBGF-8, FGF8, AIGF
Uniprot:P55075
Reactivity:Human
Range:15.6-1000 pg/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
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
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.
ELISA
Human FGF8 ELISA Kit

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