Rat FGFR1 ELISA Kit (RTFI00778)

Product Name:	Rat FGFR1 ELISA Kit
Product Code:	RTFI00778
Size:	96 Assays
Target:	Rat FGFR1
Alias:	FGFR1, CD331, Flt-2, BFGFR, bFGF-R-1, CEK, FGFBR, KAL 2, KAL2, N-SAM, OGD, FGFR-1, FLGH3, FLT2H4, H2, H5, HBGFR, basic fibroblast growth factor receptor 1, CD331 antigen, Fms-like tyrosine kinase 2, fms-related tyrosine kinase 2, heparin-binding growth factor receptor, hydroxyaryl-protein kinase, Proto-oncogene c-Fgr, soluble FGFR1 variant 1, soluble FGFR1 variant 2
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat FGFR1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat FGFR1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-104	93
EDTA plasma(n=5)	85-104	96
UFH plasma(n=5)	88-103	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat FGFR1 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%	96-102%	89-104%
EDTA plasma(n=5)	92-101%	83-96%	82-95%
UFH plasma(n=5)	80-98%	80-99%	80-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q04589

UniProt Protein Function:	FGFR1: a receptor tyrosine kinase of the highly-conserved fibroblast growth factor receptor (FGFR). Binds both acidic and basic fibroblast growth factors and is involved in limb induction. Point mutations cause Pfeffer syndrome (finger and toe malformations and other skeletal errors) and dominant Kallmann syndrome 2. Stem cell leukemia lymphoma syndrome (SCLL) may be caused by a t(8;13)(p12;q12) translocation that fuses a zinc finger gene, ZNF198, to FGFR1. Various myeloproliferative disorders have been linked to translocations that fuse FGFR1 to FOP, FIM, CEP1 or the atypical kinase, Bcr. Inhibitor: SU5402. 20 isoforms of the human protein produced by alternative splicing have been described.
UniProt Protein Details:	Protein type:EC 2.7.10.1; Kinase, protein; Oncoprotein; Membrane protein, integral; Protein kinase, tyrosine (receptor); Protein kinase, TK; TK group; FGFR family Chromosomal Location of Human Ortholog: 8p11.23-p11.22 Cellular Component: integral to plasma membrane; cytoplasmic membrane-bound vesicle; plasma membrane; integral to membrane; extracellular region; cytosol; nucleus; receptor complex Molecular Function:heparin binding; identical protein binding; protein binding; fibroblast growth factor binding; protein homodimerization activity; fibroblast growth factor receptor activity; protein-tyrosine kinase activity; ATP binding Biological Process: paraxial mesoderm development; axon guidance; peptidyl-tyrosine phosphorylation; nerve growth factor receptor signaling pathway; protein amino acid autophosphorylation; cell maturation; neuron migration; negative regulation of transcription from RNA polymerase II promoter; middle ear morphogenesis; protein amino acid phosphorylation; positive regulation of MAP kinase activity; sensory perception of sound; positive regulation of MAPKKK cascade; ureteric bud development; induction of an organ; regulation of cell differentiation; midbrain development; positive regulation of cell proliferation; positive regulation of mesenchymal cell proliferation; chondrocyte differentiation; angiogenesis; skeletal development; embryonic limb morphogenesis; positive regulation of cardiac muscle cell proliferation; epidermal growth factor receptor signaling pathway; inner ear morphogenesis; cell migration; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; chordate embryonic development; transcription, DNA-dependent; in utero embryonic development; outer ear morphogenesis; MAPKKK cascade; positive regulation of cell cycle; positive regulation of phosphoinositide 3-kinase cascade; neuroblast division in the ventricular zone; mesenchymal cell differentiation; skeletal morphogenesis; insulin receptor signaling pathway; innate immune response; auditory receptor cell development; positive regulation of neuron differentiation; regulation of lateral mesodermal cell fate specification Disease: Pfeiffer Syndrome; Hypogonadotropic Hypogonadism 2 With Or Without Anosmia; Jackson-weiss Syndrome; Trigonocephaly 1; Osteoglophonic Dysplasia
NCBI Summary:	The protein encoded by this gene is a member of the fibroblast growth factor receptor (FGFR) 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 binds both acidic and basic fibroblast growth factors and is involved in limb induction. Mutations in this gene have been associated with Pfeiffer syndrome, Jackson-Weiss syndrome, Antley-Bixler syndrome, osteoglophonic dysplasia, and autosomal dominant Kallmann syndrome 2. Chromosomal aberrations involving this gene are associated with stem cell myeloproliferative disorder and stem cell leukemia lymphoma syndrome. Alternatively spliced variants which encode different protein isoforms have been described; however, not all variants have been fully characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	Q04589
NCBI GenInfo Identifier:	291327489
NCBI Gene ID:	2260
NCBI Accession:	NP_001167534.1
UniProt Secondary Accession:	Q04589,Q90Z00, P22182, Q04589, P16092, P21804,
UniProt Related Accession:	P11362
Molecular Weight:
NCBI Full Name:	fibroblast growth factor receptor 1 isoform 10
NCBI Synonym Full Names:	fibroblast growth factor receptor 1
NCBI Official Symbol:	FGFR1
NCBI Official Synonym Symbols:	CEK; FLG; HH2; OGD; ECCL; FLT2; KAL2; BFGFR; CD331; FGFBR; FLT-2; HBGFR; N-SAM; FGFR-1; HRTFDS; bFGF-R-1
NCBI Protein Information:	fibroblast growth factor receptor 1
UniProt Protein Name:	Fibroblast growth factor receptor 1
UniProt Synonym Protein Names:	Basic fibroblast growth factor receptor 1; BFGFR; bFGF-R-1; Fms-like tyrosine kinase 2; FLT-2; N-sam; Proto-oncogene c-Fgr
Protein Family:	FGFR1 oncogene partner
UniProt Gene Name:	FGFR1
UniProt Entry Name:	FGFR1_HUMAN

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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.