Product Name:	Human FGFR2 / CD332 / FGF Receptor 2 ELISA Kit
Product Code:	HUFI02459
Size:	96 Assays
Alias:	FGFR2, CD332, BFR-1, CD332 antigen, CFD1, craniofacial dysostosis 1, EC 2.7.10, EC 2.7.10.1, fibroblast growth factor receptor 2, FLJ98662, Jackson-Weiss syndrome, JWS, KeRatinocyte growth factor receptorreceptor like 14, KSAM, K-sam, soluble FGFR4 variant 4
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human FGFR2 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human FGFR2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human FGFR2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-99	94
EDTA plasma(n=5)	88-100	93
UFH plasma(n=5)	89-102	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human FGFR2 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)	85-102%	86-105%	85-103%
EDTA plasma(n=5)	86-95%	83-99%	84-101%
UFH plasma(n=5)	85-100%	81-97%	81-98%

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

P21802

UniProt Protein Function:	FGFR2: a receptor tyrosine kinase of the highly-conserved FGFR family that binds fibroblast growth factor (FGF). Mutations are associated with many craniosynostotic syndromes and bone malformations. Mutations cause syndromes with defects in facial and limb development, including Crouzon syndrome, Beare-Stevenson cutis gyrata syndrome, Pfeiffer syndrome, Apert syndrome, and Jackson-Weiss syndrome. Somatic mutations seen in gastric cancer. Amplified in gastric, breast and some B cell cancers, but deleted in glioblastoma Twenty splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:EC 2.7.10.1; FGFR family; Kinase, protein; Membrane protein, integral; Oncoprotein; Protein kinase, TK; Protein kinase, tyrosine (receptor); TK group Chromosomal Location of Human Ortholog: 10q26.13 Cellular Component: cell cortex; cell surface; cytoplasm; excitatory synapse; extracellular matrix; integral to plasma membrane; intracellular membrane-bound organelle; nucleoplasm; nucleus; plasma membrane Molecular Function:1-phosphatidylinositol-3-kinase activity; fibroblast growth factor binding; fibroblast growth factor receptor activity; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; protein binding; protein homodimerization activity; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity Biological Process: alveolus development; angiogenesis; axonogenesis; bone mineralization; branching morphogenesis of a nerve; cell fate commitment; cell-cell signaling; embryonic cranial skeleton morphogenesis; embryonic digestive tract morphogenesis; embryonic organ development; embryonic organ morphogenesis; embryonic pattern specification; epidermis morphogenesis; epithelial cell differentiation; fibroblast growth factor receptor signaling pathway; gland morphogenesis; gut development; hair follicle morphogenesis; in utero embryonic development; inner ear morphogenesis; lacrimal gland development; limb bud formation; lung development; MAPKKK cascade; mesenchymal cell differentiation; midbrain development; morphogenesis of embryonic epithelium; multicellular organism growth; negative regulation of transcription from RNA polymerase II promoter; neuroblast division in the ventricular zone; odontogenesis; orbitofrontal cortex development; organ growth; organ morphogenesis; otic vesicle formation; peptidyl-tyrosine phosphorylation; phosphoinositide-mediated signaling; positive regulation of cardiac muscle cell proliferation; positive regulation of cell cycle; positive regulation of cell division; positive regulation of cell proliferation; positive regulation of epithelial cell proliferation; positive regulation of MAPKKK cascade; positive regulation of mesenchymal cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of Wnt receptor signaling pathway; post-embryonic development; protein amino acid autophosphorylation; pyramidal neuron development; regulation of cell fate commitment; regulation of fibroblast growth factor receptor signaling pathway; regulation of multicellular organism growth; regulation of osteoblast differentiation; regulation of osteoblast proliferation; regulation of phosphoinositide 3-kinase cascade; regulation of smooth muscle cell differentiation; regulation of smoothened signaling pathway; reproductive structure development; skeletal morphogenesis; ureteric bud development; ventricular cardiac muscle morphogenesis Disease: Antley-bixler Syndrome Without Genital Anomalies Or Disordered Steroidogenesis; Apert Syndrome; Beare-stevenson Cutis Gyrata Syndrome; Bent Bone Dysplasia Syndrome; Crouzon Syndrome; Gastric Cancer; Jackson-weiss Syndrome; Lacrimoauriculodentodigital Syndrome; Pfeiffer Syndrome; Saethre-chotzen Syndrome; Scaphocephaly, Maxillary Retrusion, And Mental Retardation
NCBI Summary:	The protein encoded by this gene is a member of the fibroblast growth factor receptor family, where amino acid sequence is highly conserved between members and throughout evolution. FGFR family members differ from one another in their ligand affinities and tissue distribution. A full-length representative protein consists of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This particular family member is a high-affinity receptor for acidic, basic and/or keratinocyte growth factor, depending on the isoform. Mutations in this gene are associated with Crouzon syndrome, Pfeiffer syndrome, Craniosynostosis, Apert syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrata syndrome, Saethre-Chotzen syndrome, and syndromic craniosynostosis. Multiple alternatively spliced transcript variants encoding different isoforms have been noted for this gene. [provided by RefSeq, Jan 2009]
UniProt Code:	P21802
NCBI GenInfo Identifier:	120049
NCBI Gene ID:	2263
NCBI Accession:	P21802.1
UniProt Secondary Accession:	P21802,P18443, Q01742, Q12922, Q14300, Q14301, Q14302 Q14303, Q14304, B4DFC2, E7EVR6, E9PCR0,
UniProt Related Accession:	P21802
Molecular Weight:	79,833 Da
NCBI Full Name:	Fibroblast growth factor receptor 2
NCBI Synonym Full Names:	fibroblast growth factor receptor 2
NCBI Official Symbol:	FGFR2
NCBI Official Synonym Symbols:	BEK; JWS; BBDS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1; CD332; K-SAM
NCBI Protein Information:	fibroblast growth factor receptor 2
UniProt Protein Name:	Fibroblast growth factor receptor 2
UniProt Synonym Protein Names:	K-sam; KGFR; Keratinocyte growth factor receptor; CD_antigen: CD332
Protein Family:	Tumor necrosis factor receptor superfamily
UniProt Gene Name:	FGFR2

*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 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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Human FGFR2 / CD332 / FGF Receptor 2 ELISA Kit

Description