Overview

  • Product name
  • Description
    Rabbit polyclonal to FMRP
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-FoFr, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 600 to the C-terminus of Human FMRP.

    Read Abcam's proprietary immunogen policy

  • Positive control
    • This antibody gave a positive signal in HeLa whole cell lysate and HeLa nuclear lysate. This antibody gave a positive result when used in the following formaldehyde fixed cell lines: SKNSH.
  • General notes
    ab27455 does not recognise endogenous FMRP (expected size 71 kDa) in human testes lysate, which may be due to low expression levels of FMRP.

Properties

Applications

Our Abpromise guarantee covers the use of ab27455 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
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 85 kDa (predicted molecular weight: 71 kDa).
IHC-FoFr Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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

  • All lanes : Anti-FMRP antibody (ab27455) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

    Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution

    Predicted band size: 71 kDa
    Observed band size: 85 kDa
    why is the actual band size different from the predicted?



    ab27455 detects a band around 85 kDa in Hela and Hela Nuclear lysate, which is consistent with bands seen by other researchers using FMRP antibodies.
  • IHC image of FMRP staining in human cerebral cortex FFPE 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 ab27455, 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.
  • ab27455 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 ab27455 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.

References

This product has been referenced in:
  • Guo J  et al. Chromatin and Single-Cell RNA-Seq Profiling Reveal Dynamic Signaling and Metabolic Transitions during Human Spermatogonial Stem Cell Development. Cell Stem Cell 21:533-546.e6 (2017). Read more (PubMed: 28985528) »
  • Say E  et al. A functional requirement for PAK1 binding to the KH(2) domain of the fragile X protein-related FXR1. Mol Cell 38:236-49 (2010). WB ; Human . Read more (PubMed: 20417602) »
See all 4 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer


The lot currently in stock is 943249, we have 17 units left.

Read More

Question

Dear technical support team:

This customer has purchased ab27455 (Anti-FMRP antibody) and has conducted the wb several times with human sample. The results show wrong band size, therefore, she would like to ask for your help to modify her experiment step, could you please offer any suggestion to improve her result?

I attached the image in this letter and her experiment step as follow:



1. Order details:


Batch number:

Abcam product code: ab27455

Antibody storage conditions (temperature/reconstitution etc) :10ul/tube store at -20℃




2. Please describe the problem (high background, wrong band size, more bands, no band etc).

Wrong band size



3. On what material are you testing the antibody in WB?

· Species: Human

· What’s cell line or tissue: Huh7 & Hela cell line

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein:

3. The lysate


How much protein was loaded: First :20ug Second:20ug &30ug

What lysis buffer was used: 50mMTris-HCl,pH7.4, 150mM NaCl


1mM EDTA, 1%Triton x-100,


What protease inhibitors were used: Roche protease inhibitor (25X→1X)

What loading buffer was used: 5X sample buffer


(250mm Tris-HCl, 10%SDS, 30%glycerol, 5%b-mercaptoethanol,0.1%Bromophenol Blue)


Phosphatase inhibitors :YES

Did you heat the samples: temperature and time: 95℃ 5mins


4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: non reducing gel

Reducing agent:no

Gel percentage : 12.5%


Transfer conditions: (Type of membrane, Protein transfer verified): Nitrocellulose paper, 100V 80mins

5. Blocking conditions


Buffer:TTBS

Blocking agent: milk, BSA, serum, what percentage:


First exp.- 7% non-fat milk Second exp.- BlockPRO blocking buffer


Incubation time: 1hrs

Incubation temperature: room tempture


6. Primary Antibody


Species:Rabbit

Reacts against: Human


· At what dilution(s) have you tested this antibody:1:1000

· What dilution buffer was used: TTBS

· Incubation time: overnight

· Incubation temperature: 4℃

· What washing steps were done: TTBS wash 5mins, total 3 times

7. Secondary Antibody


Species:Goat

Reacts against: Rabbit

At what dilution(s) have you tested this antibody: 1:15000

Incubation time: 1hr

Wash steps: TTBS wash 5mins, total 3 times

Fluorochrome or enzyme conjugate:HRP

Do you know whether the problems you are experiencing come from the secondary?


8. Detection method
ECl, ECl+, other detection method:ECL

9. Did you apply positive and negative controls along with the samples? Please specify.

10. Optimization attempts

· How many times have you tried the Western? 2 times

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? Blocking condition and protein loading



Could you please help this customer to solve the problem?

Thanks for your kindly help

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab27455 Anti-FMRP antibody. I would also appreciate if you can confirm some further details:

The human FMRP has six isoforms, and all of them contain the same C-terminus from which the antibody's immunogen is derived from.

Almost allof our antibodies are developed to work under reduced- denaturing conditions. I would strongly advise to run the gel under reducing conditions to assure that the epitope can be found by the antibody.

It may be worth a to perform a nuclear fraction protocol to "clean up" the blot. The effect can be seen in the WB image the datasheet of ab27455, where a whole cell lysate and a nuclear fraction lysate have been run in comparison.

For a better saturation of the membrane, I would recommend a blocking buffer containing 3% BSA for 2 hours at room temperature. BSA can sometimes give better results than milk.

Increase the primary antibodydilution to 1:2000 to increase the specificity of the antibody.

As this is not avery lightprotein, I would suggestto run agel with a decreased gel concentration. This should facilitate the proteins migration in the gel and usually gives a better resolution of the gel.




Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Specification
Brain

Dr. Sophie Pezet

Verified customer

Submitted Jul 29 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T)
Specification
HEK293T
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C

Abcam user community

Verified customer

Submitted May 20 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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