Overview

  • Product name
    Anti-FMRP (phospho S499) antibody
    See all FMRP primary antibodies
  • Description
    Rabbit polyclonal to FMRP (phospho S499)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, ELISAmore details
  • Species reactivity
    Reacts with: Human, Zebrafish
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 450 - 550 of Human FMRP.

    Read Abcam's proprietary immunogen policy (Peptide available as ab48658.)

  • Positive control
    • This antibody gave a positive signal in both HeLa (Human epithelial carcinoma cell line) and HeLa Nuclear Lysate as well as Mouse brain tissue lysate. It also gave a positive signal in FFPE human cerebral cortex tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab48127 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 before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 71 kDa).
ICC/IF Use at an assay dependent concentration. PubMed: 24658146
ELISA Use at an assay dependent concentration.

Target

  • Function
    Translation repressor. Component of the CYFIP1-EIF4E-FMR1 complex which binds to the mRNA cap and mediates translational repression. In the CYFIP1-EIF4E-FMR1 complex this subunit mediates translation repression (By similarity). RNA-binding protein that plays a role in intracellular RNA transport and in the regulation of translation of target mRNAs. Associated with polysomes. May play a role in the transport of mRNA from the nucleus to the cytoplasm. Binds strongly to poly(G), binds moderately to poly(U) but shows very little binding to poly(A) or poly(C).
  • Tissue specificity
    Highest levels found in neurons, brain, testis, placenta and lymphocytes. Also expressed in epithelial tissues and at very low levels in glial cells.
  • Involvement in disease
    Defects in FMR1 are the cause of fragile X syndrome (FRAX) [MIM:300624]. Fragile X syndrome is a common genetic disease (has a prevalence of one in every 2000 children) which is characterized by moderate to severe mental retardation, macroorchidism (enlargement of the testicles), large ears, prominent jaw, and high-pitched, jocular speech. The defect in most fragile X syndrome patients results from an amplification of a CGG repeat region which is directly in front of the coding region.
    Defects in FMR1 are the cause of fragile X tremor/ataxia syndrome (FXTAS) [MIM:300623]. In FXTAS, the expanded repeats range in size from 55 to 200 repeats and are referred to as 'premutations'. Full repeat expansions with greater than 200 repeats results in fragile X mental retardation syndrome [MIM:300624]. Carriers of the premutation typically do not show the full fragile X syndrome phenotype, but comprise a subgroup that may have some physical features of fragile X syndrome or mild cognitive and emotional problems.
    Defects in FMR1 are the cause of premature ovarian failure syndrome type 1 (POF1) [MIM:311360]. An ovarian disorder defined as the cessation of ovarian function under the age of 40 years. It is characterized by oligomenorrhea or amenorrhea, in the presence of elevated levels of serum gonadotropins and low estradiol.
  • Sequence similarities
    Belongs to the FMR1 family.
    Contains 2 KH domains.
  • Post-translational
    modifications
    Phosphorylated on several serine residues.
  • Cellular localization
    Cytoplasm. Nucleus > nucleolus.
  • Information by UniProt
  • Database links
  • Alternative names
    • FMR 1 antibody
    • Fmr1 antibody
    • Fmr1 gene antibody
    • FMR1_HUMAN antibody
    • FMRP antibody
    • Fragile X mental retardation 1 antibody
    • Fragile X mental retardation 1 protein antibody
    • Fragile X mental retardation protein 1 antibody
    • Fragile X mental retardation protein antibody
    • fragile X mental retardation syndrome-related protein 1 antibody
    • fragile X mental retardation, autosomal homolog 1 antibody
    • FRAXA antibody
    • fxr1 antibody
    • MGC87458 antibody
    • POF antibody
    • POF1 antibody
    • Protein FMR-1 antibody
    • Protein FMR1 antibody
    • wu:fb16f11 antibody
    • wu:fd18c10 antibody
    • zgc:66226 antibody
    see all

Images

  • Serially diluted ab48127 was bound to immobilised phospho (ab48658) - or control (unmodified) peptides (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50,000 times) and signal was developed with TMB substrate

  • All lanes : Anti-FMRP (phospho S499) antibody (ab48127) at 2 µg/ml

    Lane 1 : Human brain tissue lysate - total protein (ab29466)
    Lane 2 : Mouse Brain Tissue Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 71 kDa
    Additional bands at: 71 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 8 minutes
  • IHC image of FMRP (phospho S499) staining in human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 48127, 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.
  • All lanes : Anti-FMRP (phospho S499) antibody (ab48127) at 0.25 µg/ml

    Lane 1 : Marker
    Lane 2 : Zebrafish brain homogenate at 20 µg
    Lane 3 : Zebrafish heart homogenate at 20 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 71 kDa
    Observed band size: 71 kDa


    Exposure time: 5 minutes

References

This product has been referenced in:
  • Lechpammer M  et al. Dysregulation of FMRP/mTOR Signaling Cascade in Hypoxic-Ischemic Injury of Premature Human Brain. J Child Neurol 31:426-32 (2016). Read more (PubMed: 26239490) »
  • Spencer KB  et al. FMRP Mediates Chronic Ethanol-Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus. Alcohol Clin Exp Res 40:1251-61 (2016). Read more (PubMed: 27147118) »
See all 7 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Answer

Thank you very much for your reply.

I'll be happy to send the blocking peptide. Do you have an order or PO number?

I look forward to hearing from you. Please let me know if there is anything else you need from me at this time.

Read More
Application
Immunoprecipitation
Sample
Zebrafish Tissue lysate - whole (brain)
Total protein in input
1000 µg
Immuno-precipitation step
Protein A/G
Specification
brain

Abcam user community

Verified customer

Submitted Jul 16 2015

Application
Western blot
Sample
Zebrafish Tissue lysate - whole (brain)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C

Abcam user community

Verified customer

Submitted Jul 16 2015

Question
Answer

Thank you very much for your replies today.

I'm sending the peptide free of charge on the order ***, which should arrive within the next two days.

Please keep me updated about any new results using the blocking peptide, and let me know if there is anything else that we can do for you.

Read More

Answer

Thank you so much for sending the additional information and for your patience. I'm in the midst of a literature search but I will be back in touch this afternoon. Please let me know if you have any questions or need anything else in the meantime.

Read More

Answer

Thank you for your call yesterday and for sending the images.

I have a couple of additional questions. There are three blots showing results with hippocampus tissue- is this all tissue from the same animal, or are they different animals? Is the cortex tissue from the same animal? Are these blots all taken with the same protocol and exposure time?

We haven't tested this antibody with rat tissue, but we have seen bands at 50 and 75 in testing with human and mouse lysates, and both these bands were blocked by the immunogen peptide. So, I think that these bands are specific. I haven't seen any other data showing a band around 200 kDa so this is most likely non-specific. Several isoforms ofFMRP have been identified in mouse and human, less so in rat, but I do believe these are different isoforms. I've linked the UniProt pages below for reference.

http://www.uniprot.org/uniprot/P35922

http://www.uniprot.org/uniprot/Q80WE1

http://www.uniprot.org/uniprot/Q06787

I'll have to look through the literature to see what isoforms might be expressed in rat brain tissue, as we haven't tested any of our FMRP antibodies with this kind of tissue. A good negative control might help distinguish specific bands from background. You could use the immunogen peptide ab48658 in a blocking assay, which should help identify the specific bands.

I'll be back in touch on Monday after reviewing the literature and hopefully getting some more information for you. Please let me know if you have any questions or need anything else in the meantime.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
No

Dr. Sophie Pezet

Verified customer

Submitted Jun 08 2012

Answer

DISCOUNT CODE: *************
Expiration date: ***************
I am very pleased to hear you would like to accept our offer and test ab48127 in mouse. This code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.
Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.
Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.
The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Question
Answer

DISCOUNT CODE: *********

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Answer

Thank you for your review of ab48127, anti-FMRP (phospho S499), which I just published. The western blot result is disappointing, given the lack of a strong signal at the expected molecular weight of 71 kDa. I do not think the bands at 135 kDa or 125 kDa are FMRP. Have you stained blots of these samples for total FMR1, in addition to this phosphorylation? I am curious if an immunostained blot for total FMR1 shows the protein at 71 kDa. One suggestion that may help develop the signal at 71 kDa is to avoid using milk in your antibody diluent. I am not sure if you did, but I see that the blocking solution is 5% milk. Milk in the blocking solution does not appear to be a problem, since there is no overall dark background, which would indicate that the antibody cross-reacts with a phospho-protein in the milk itself. My only other suggestion would be to ensure that you include a phosphatase inhibitor in your lysis buffer. If you did this, and feel that your protocol is correct and that the samples are intact and phosphorylated, I would like to offer you a free replacement or credit or refund for this antibody. Please contact me if you are interested.

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1-10 of 13 Abreviews or Q&A

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