Overview

  • Product name

    Anti-FGFR1 (phospho Y654) antibody
    See all FGFR1 primary antibodies
  • Description

    Rabbit polyclonal to FGFR1 (phospho Y654)
  • Host species

    Rabbit
  • Specificity

    Binds human and mouse FGFR1 only when phosphorylated at tyrosine 654 and rat FGFR1 only when phosphorylated at tyrosine 561.
  • Tested applications

    Suitable for: IHC-P, ICC/IF, WB, ELISAmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human FGFR1. Synthetic phosphopeptide (Human) from around the phosphorylation site of tyrosine 654 (DYYPKK)
    Database link: P11362

  • Positive control

    • WB: 293 and HeLa cell lysates treated with EGF, 293 cell extract treated with insulin. IHC-P: Human breast adenocarcinoma. ICC/IF: SKNSH cells. COS7 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab59194 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 5 µg/ml.
WB 1/500 - 1/1000. Detects a band of approximately 117 kDa (predicted molecular weight: 92 kDa).
ELISA 1/20000.

Target

  • Function

    Receptor for basic fibroblast growth factor. Receptor for FGF23 in the presence of KL (By similarity). A shorter form of the receptor could be a receptor for FGF1 (aFGF).
  • Tissue specificity

    Detected in astrocytoma, neuroblastoma and adrenal cortex cell lines. Some isoforms are detected in foreskin fibroblast cell lines, however isoform 17, isoform 18 and isoform 19 are not detected in these cells.
  • Involvement in disease

    Defects in FGFR1 are a cause of Pfeiffer syndrome (PS) [MIM:101600]; also known as acrocephalosyndactyly type V (ACS5). PS is characterized by craniosynostosis (premature fusion of the skull sutures) with deviation and enlargement of the thumbs and great toes, brachymesophalangy, with phalangeal ankylosis and a varying degree of soft tissue syndactyly.
    Defects in FGFR1 are a cause of idiopathic hypogonadotropic hypogonadism (IHH) [MIM:146110]. 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.
    Defects in FGFR1 are the cause of Kallmann syndrome type 2 (KAL2) [MIM:147950]; also known as 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 cases, midline cranial anomalies (cleft lip/palate and imperfect fusion) are present and anosmia may be absent or inconspicuous.
    Defects in FGFR1 are the cause of osteoglophonic dysplasia (OGD) [MIM:166250]; also known as osteoglophonic dwarfism. OGD is characterized by craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as by rhizomelic dwarfism and nonossifying bone lesions. Inheritance is autosomal dominant.
    Defects in FGFR1 are the cause of trigonocephaly non-syndromic (TRICEPH) [MIM:190440]; also known as metopic craniosynostosis. The term trigonocephaly describes the typical keel-shaped deformation of the forehead resulting from premature fusion of the frontal suture. Trigonocephaly may occur also as a part of a syndrome.
    Note=A chromosomal aberration involving FGFR1 may be a cause of stem cell leukemia lymphoma syndrome (SCLL). Translocation t(8;13)(p11;q12) with ZMYM2. SCLL usually presents as lymphoblastic lymphoma in association with a myeloproliferative disorder, often accompanied by pronounced peripheral eosinophilia and/or prominent eosinophilic infiltrates in the affected bone marrow.
    Note=A chromosomal aberration involving FGFR1 may be a cause of stem cell myeloproliferative disorder (MPD). Translocation t(6;8)(q27;p11) with FGFR1OP. Insertion ins(12;8)(p11;p11p22) with FGFR1OP2. MPD is characterized by myeloid hyperplasia, eosinophilia and T-cell or B-cell lymphoblastic lymphoma. In general it progresses to acute myeloid leukemia. The fusion proteins FGFR1OP2-FGFR1, FGFR1OP-FGFR1 or FGFR1-FGFR1OP may exhibit constitutive kinase activity and be responsible for the transforming activity.
    Note=A chromosomal aberration involving FGFR1 may be a cause of stem cell myeloproliferative disorder (MPD). Translocation t(8;9)(p12;q33) with CEP110. MPD is characterized by myeloid hyperplasia, eosinophilia and T-cell or B-cell lymphoblastic lymphoma. In general it progresses to acute myeloid leukemia. The fusion protein CEP110-FGFR1 is found in the cytoplasm, exhibits constitutive kinase activity and may be responsible for the transforming activity.
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. Fibroblast growth factor receptor subfamily.
    Contains 3 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications

    Binding of FGF1 and heparin promotes autophosphorylation on tyrosine residues and activation of the receptor.
  • Cellular localization

    Membrane. Nucleus. Cytoplasm. Cytoplasmic vesicle
  • Information by UniProt
  • Database links

  • Alternative names

    • Basic fibroblast growth factor receptor 1 antibody
    • bFGF-R-1 antibody
    • BFGFR antibody
    • CD331 antibody
    • CEK antibody
    • FGFBR antibody
    • FGFR 1 antibody
    • FGFR-1 antibody
    • FGFR1 antibody
    • FGFR1/PLAG1 fusion antibody
    • FGFR1_HUMAN antibody
    • fibroblast growth factor receptor 1 antibody
    • FLG antibody
    • FLT-2 antibody
    • FLT2 antibody
    • Fms-like gene antibody
    • Fms-like tyrosine kinase 2 antibody
    • fms-related tyrosine kinase 2 antibody
    • HBGFR antibody
    • heparin-binding growth factor receptor antibody
    • HH2 antibody
    • HRTFDS antibody
    • hydroxyaryl-protein kinase antibody
    • KAL2 antibody
    • N-SAM antibody
    • OGD antibody
    • Proto-oncogene c-Fgr antibody
    see all

Images

  • All lanes : Anti-FGFR1 (phospho Y654) antibody (ab59194) at 1/1000 dilution

    Lane 1 : 293 untreated lysates
    Lane 2 : 293 treated with 200 ng/mL EGF for 30 minutes
    Lane 3 : HeLa untreated lysates
    Lane 4 : HeLa treated with 200 ng/mL EGF for 30 minutes

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG (H+L) HRP at 1/10000 dilution

    Predicted band size: 92 kDa
    Observed band size: 156 kDa
    why is the actual band size different from the predicted?



    Loading control: Beta Actin

  • IHC image of FGFR1 (phospho Y654)  staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab59194, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab59194 stained SKNSH cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab59194, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  • All lanes : Anti-FGFR1 (phospho Y654) antibody (ab59194) at 1/500 dilution

    Lane 1 : 293 cell extract treated with insulin (0.01U/ml, 15 mins)
    Lane 2 : 293 cell extract treated with insulin (0.01U/ml, 15 mins) with immunizing phosphopeptide

    Predicted band size: 92 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?

  • Immunofluorescent analysis of COS7 cells labeling FGFR1 (phospho-Tyr654 with ab59194 at 1:100. The image on the right is blocked with the phosphopeptide prior to imunnoflurescent labeling.

References

This product has been referenced in:

  • Murahashi Y  et al. Multi-layered PLLA-nanosheets loaded with FGF-2 induce robust bone regeneration with controlled release in critical-sized mouse femoral defects. Acta Biomater 85:172-179 (2019). Read more (PubMed: 30583110) »
  • Lee KW  et al. FGF11 influences 3T3-L1 preadipocyte differentiation by modulating the expression of PPAR? regulators. FEBS Open Bio 9:769-780 (2019). Read more (PubMed: 30984550) »
See all 14 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Thank you for contacting us.

I have conducted a search through our catalogue for antibodies against pFGFR1 and pFGFR4 which do no cross-react with the other FGFRs.

Ab59180 is specific against FGFR1 when it is phosphorilated at Tyr766. Similarly, ab59194 is specific against FGFR1 when it is phosphorilated at Tyr654, while ab52165 is specific against FGFR1 when it is phosphorilated at Tyr154.

These three antibodies do not bind to non-phosphorilated proteins. If you would send me the accession numbers or the exact protein sequences of other FGFRs that you are concerned with cross reactivity, I can align them with our immunogen sequence for sequence homology and possible cross reactivity.

We do have other alternatives (ab111124 and ab134043) against pFGFR1. However, these antibodies may also detect FGFR2, FGFR3 and FGFR4 phosphorilated at the equivalent sites. Although the antibodies have not been tested to determine if it detects these other endogenous forms, based on sequence homology reactivity would be expected.

Unfortunately, all the https://www.abcam.com/Search?Keywords=FGFR4+&fReset=1&pt=1&source=TopSearch&btnSearch=Search are against non phosphorilated forms, so I am afraid they would not fit your requirements.

Please do not hesitate to let me know whether you require a proforma, or any further assistance.

Read More
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (hair follicle)
Permeabilization
Yes - PBST 0.05% (tween)
Specification
hair follicle
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 12 2018

Question
Answer

For diluting these antibodies for western blotting, we recommend PBS, 0.1% Triton X-100, with 1% BSA.

Read More

Question
Answer

Thank you for contacting us.

I have received the following information from the lab:

1) This antibody reacts with target strongly when target is phosphorylated only on Y654 (phospho-Y654).
2) This antibody has weak reaction with target when both Y653 and Y654 sites are phosphorylated (phospho-Y653-Y654).
3) This antibody does not react with target when target is phosphorylated only on Y653.

We tested with three phospho-peptides: phospho-Y653-peptide; phospho-Y654-peptide; and phospho-Y653-Y654-peptide (double phospho sites).

The phospho-Y654-peptide blocked this antibody reaction, completely.
The phospho-Y653-Y654-peptide (double phosphos sites) blocked the reaction, partially.
The phospho-Y653-peptide did not block the reaction.



Here is also some more information about 1) our Apromise guarantee (for tested applications/species) and 2) our testing discount program (for UNTESTED application/species).

1) We guarantee all our products for tested species and applications as listed on the datasheet. The guarantee is valid for 6 months after a purchase. For more details about our Abpromise guarantee please check this link: https://www.abcam.com/abpromise.

2) For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:
The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab59194. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

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Answer

Thank you for contacting us. Indeed, crossreactivity is always a very important information for an antibody. Sequence alignment and experimental data can help to predict/determine this. We receive these antibodies from a collaborating laboratory. I have asked the laboratory to know how the specificity has been determined and whether crossreactivity has experimentally been assessed. The laboratory had not assessed this crossreactivity experimentally. They have run a blast with the exact immunogen sequences (to which I have no access) and does on that basis exclude any possible crossreaction with other proteins than FGFR1. The regions from which the respective peptide immunogen of the antibodies are ab52165 (Tyr154), ab59180 (Tyr 766) and ab59194 (Tyr654). Please find here the sequence alignment for FGFR1 with FGFR2: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-091151-0127-47989414-oy For predicted crossreactivity with FGFR2: Comparison of the sequence with FGFR3 and FGFR1: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-103134-0638-15807362-pg Sequence alignment of FGFR1 with FGFR4: http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110907-103350-0499-7232840-pg The sequence around Tyr154 has deletions in the FGFR2 and FGFR3 sequence when compared to the sequence of FGFR1, resulting therefore in a very poor alignment of that region. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us. The protocol is as follows: a. 293 cells were plated on flask or dishes containing growth medium (basic medium containing 10% serum). b. When the cells were at the log phase and 80-90% confluent, the culture media was drained. Wash cells with fresh basic media (no serum) once. c. Add fresh basic media to the vessels and starve the cells for 18-24 hours. d. Drain the cultured medium, then add fresh basic medium (about 4ml per T75 flask) to the vessels. e. Add insulin to the medium at 0.01U/ml. f. Cells were incubated at 37°C for 15 minutes in a CO2 incubator and then were used to prepare cell lysis. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry and your interest in our products. I have discussed your enquiry with my colleagues in the Lab and we think the antibody can detect both endogenous FGFR1 (phospho Y654) and the induced/stimulated FGFR1 at this modification site. We have only tested the antibody in 293 cells treated with insulin. I hope this will be usefulr for you.

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