Overview

  • Product name
  • Description
    Rabbit polyclonal to CPEB1
  • Host species
    Rabbit
  • Specificity
    Detects recombinant mouse CPEB1.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse
    Predicted to work with: Rat, Human, Xenopus laevis
  • Immunogen

    Synthetic peptide corresponding to Human CPEB1 aa 545-562.
    Sequence:

    HSMEGLRHHSPLMRNQKN


    (Peptide available as ab4988)

Properties

Applications

Our Abpromise guarantee covers the use of ab3465 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 1/50 - 1/500.
ICC/IF 1/50 - 1/500.
IP Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).Can be blocked with Human CPEB1 peptide (ab4988).

Target

  • Function
    Sequence-specific RNA-binding protein that regulates mRNA cytoplasmic polyadenylation and translation initiation during oocyte maturation, early development and at postsynapse sites of neurons. Binds to the cytoplasmic polyadenylation element (CPE), an uridine-rich sequence element (consensus sequence 5'-UUUUUAU-3') within the mRNA 3'-UTR. In absence of phosphorylation and in association with TACC3 is also involved as a repressor of translation of CPE-containing mRNA; a repression that is relieved by phosphorylation or degradation (By similarity). Involved in the transport of CPE-containing mRNA to dendrites; those mRNAs may be transported to dendrites in a translationally dormant form and translationally activated at synapses (By similarity). Its interaction with APLP1 promotes local CPE-containing mRNA polyadenylation and translation activation (By similarity). Induces the assembly of stress granules in the absence of stress.
  • Tissue specificity
    Isoform 1 is expressed in immature oocytes, ovary, brain and heart. Isoform 2 is expressed in brain and heart. Isoform 3 and isoform 4 are expressed in brain. Expressed in breast tumors and several tumor cell lines.
  • Sequence similarities
    Belongs to the RRM CPEB family.
    Contains 2 RRM (RNA recognition motif) domains.
  • Domain
    The 2 RRM domains and the C-terminal region mediate interaction with CPE-containing RNA.
  • Post-translational
    modifications
    Phosphorylated on serine/threonine residues by AURKA/STK6 within positions 166 and 197. Phosphorylation and dephosphorylation on Thr-172 regulates cytoplasmic polyadenylation and translation of CPE-containing mRNAs. Phosphorylation on Thr-172 by AURKA/STK6 and CAMK2A activates CPEB1. Phosphorylation on Thr-172 may be promoted by APLP1. Phosphorylation increases binding to RNA.
  • Cellular localization
    Cytoplasm > P-body. Cytoplasmic granule. Cell junction > synapse. Membrane. Cell junction > synapse > postsynaptic cell membrane > postsynaptic density. Cell projection > dendrite. Also found in stress granules. Recruited to stress granules (SGs) upon arsenite treatment. In dendrites (By similarity). Localizes in synaptosomes at dendritic synapses of neurons (By similarity). Strongly enriched in postsynaptic density (PSD) fractions (By similarity). Transported into dendrites in a microtubule-dependent fashion and colocalizes in mRNA-containing particles with TACC3, dynein and kinesin (By similarity). Membrane-associated (By similarity). Colocalizes at excitatory synapses with members of the polyadenylation and translation complex factors (CPSF, APLP1, TACC3, AURKA/STK6, SYP, etc.) including CPE-containing RNAs.
  • Information by UniProt
  • Database links
  • Alternative names
    • CEBP antibody
    • CPE binding protein 1 antibody
    • CPE BP1 antibody
    • CPE-binding protein 1 antibody
    • CPE-BP1 antibody
    • CPEB 1 antibody
    • CPEB antibody
    • CPEB-1 antibody
    • CPEB1 antibody
    • CPEB1 protein antibody
    • CPEB1_HUMAN antibody
    • Cytoplasmic polyadenylation binding protein 1 antibody
    • Cytoplasmic polyadenylation element binding protein antibody
    • Cytoplasmic polyadenylation element binding protein 1 antibody
    • Cytoplasmic polyadenylation element-binding protein 1 antibody
    • FLJ13203 antibody
    • h CEBP antibody
    • h-CEBP antibody
    • hCPEB 1 antibody
    • hCPEB-1 antibody
    • MGC34136 antibody
    • MGC60106 antibody
    • mKIAA0940 antibody
    • MKIAA0940 protein antibody
    see all

Images

  • All lanes : Anti-CPEB1 antibody (ab3465) at 1/500 dilution

    Lane 1 : HeLa lysate
    Lane 2 : Human brain tissue lysate
    Lane 3 : Mouse brain tissue lysate

    Lysates/proteins at 25 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit

    Developed using the ECL technique.

    Predicted band size: 65 kDa
    Observed band size: 62 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 minute
  • Western blot detection of 1.0 ng of recombinant mouse CPEB1 using ab3465.

  • Immunohistochemistry analysis of CPEB showing staining in the cytoplasm of paraffin-embedded human hearat tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab3465 diluted in 3% BSA-PBS at a dilution of 1/50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunohistochemistry analysis of CPEB showing staining in the cytoplasm of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab3465 diluted in 3% BSA-PBS at a dilution of 1/200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunohistochemistry analysis of CPEB showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab3465 diluted in 3% BSA-PBS at a dilution of 1/100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunofluorescent analysis of CPEB (green) showing staining in the cytoplasm of U251 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab3465 in 3% BSA-PBS at a dilution of 1/100 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight®-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor® 554 (red) and nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

  • Immunofluorescent analysis of CPEB (green) showing staining in the cytoplasm of C6 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab3465 in 3% BSA-PBS at a dilution of 1/100 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight®-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

  • Immunofluorescent analysis of CPEB (green) showing staining in the cytoplasm of HeLa cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab3465 in 3% BSA-PBS at a dilution of 1/100 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight®-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor® 554 (red) and nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

References

This product has been referenced in:
  • Chen J  et al. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. Genes Dev 25:755-66 (2011). WB ; Mouse . Read more (PubMed: 21460039) »
  • Blanchard Z  et al. Geminin overexpression induces mammary tumors via suppressing cytokinesis. Oncotarget 2:1011-27 (2011). Read more (PubMed: 22184288) »
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

BATCH NUMBER 87645 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM - hardly any detectable signal irrespective of antibody-dilution and exposure time - After an exposure time of 4 minutes just an unspecific band at 25 kDa SAMPLE - activated platelet samples PRIMARY ANTIBODY - CPEB-antibody (ab3465) - Dilution: 1:1000; 1:10 000 - Diluent: 5% milk-powder in TBS-T or PBS-T - Incubation time: over night at 4?C - Wash step: three times for 15 min. with TBS-T or PBS-T respectively SECONDARY ANTIBODY - anti-rabbit from Anersham - Dilution: 1:10 000 - Diluent: milk-powder in TBS-T or PBS-T - Incubation-time: 1h at room temperature - Wash step: three times for 10 min. with TBS-T or PBS-T respectively DETECTION METHOD - ECLPlus POSITIVE AND NEGATIVE CONTROLS USED Positive controls: -human serum - mouse heart ANTIBODY STORAGE CONDITIONS - -20?C - no reconstitution SAMPLE PREPARATION - 1x SDS-Buffer - Heating procedure: 5 minutes at 95?C - Storage of the samples: at -20?C AMOUNT OF PROTEIN LOADED - approx. 25,0 ?g per lane ELECTROPHORESIS/GEL CONDITIONS - protein collection: 5% polyacrylamid-gel - protein separation: 10% Polyacrylamid-gel TRANSFER AND BLOCKING CONDITIONS - running buffer containing: 1x TG; 20% SDS; aqua dest. running time: 20 min. at 60 V; 1.30h at 120V - transfer buffer containing: methanol; 20% SDS; Na2CO3/NaHCO3-Buffer; aqua dest. transfer time: 1h at 30 mA - blocking conditions: milk powder 5% dissolved in PBS-T or TBS-T - primary antibody in milk-powder PBS-T or TBS-T solution over night - Washing: PBS-T or TBS-T respectively HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - primary antibody dilution: 1:1000 and 1:10 000 - Buffer conditions: milk-powder in PBS-T or TBS-T - Wash-step: in PBS-T or TBS-T - Exposure time: 15s, 1min, 4min, 30min up to over night exposure ADDITIONAL NOTES - In the meantime we have already obtained very good results with a CPEB-primary antibody purchased from another company which worked perfectly in our system

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing dificulty with this antibody. This is the first complaint that we have received regarding ab3465. At this point I would suggest increasing the concentration of ab3465 that you are using, try 1:500, and also decrease the stringency of your washes. You may also want to increase the concentration of your secondary antibody. Do you have an image with your results from ab3465 and the other CPEP antibody that you are using?

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Answer

Thank you for your email. Unfortunately, we are not aware of any publications that feature the use of this antibody. It seems that ab3465 may cross-react with Xenopus CPEB, but I really can't say for sure. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to test an antibody in an application for which we do not have any information, please let us know how you get on and in return we will forward a GBP10/ USD15/ EUR15 Amazon gift voucher. If you have any further questions, please contact us again.

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Answer

Thanks for your email. I'll try and answer your questionsa as fully as I can. 1) I have made enquiries about whether we can sell the MBD3 peptide. I hope this should be possible! I will let you know. 2) We would be interested in making phospho-specific antibodies to either CPEB or MBD3. Would you be interested in testing them? It takes about 4 months for us to make antibodies. Email back on my personal email if you are interested and I'll send you the details. It's ah@abcam.com Hope this helps, if not email back. I'll find out about that peptide soon for you and email if it does come on sale.

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