Overview

  • Product name
    Anti-Aurora A antibody - Centrosome Marker
    See all Aurora A primary antibodies
  • Description
    Rabbit polyclonal to Aurora A - Centrosome Marker
  • Host species
    Rabbit
  • Specificity
    ab12875 detects a single clean band of just below 50kD in Jurkat and Hela cell lysates. This band corresponds to the size of Aurora A seen using other antibodies.
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, ICCmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human Aurora A.

    Read Abcam's proprietary immunogen policy (Peptide available as ab13008.)

  • Positive control
    • Recombinant Human Aurora A protein (ab86846) can be used as a positive control in WB. Jurkat cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab12875 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
WB
ICC/IF
ICC
  • Application notes
    ICC: 1/500.
    IF: 1/500.
    IHC-P: 1/15 - 1/20. Antigen retrieval was performed using either Sodium Citrate or Tris/EDTA before commencing with the IHC staining protocol. Customer feedback suggests that a 1/15 dilution of ab12875 is recommended when using the sodium citrate antigen retreival method and a 1/20 dilution is recommended when using the Tris/EDTA antigen retrieval method.

    WB: 1/500. Detects a band of approximately 46 kDa (predicted molecular weight: 46 kDa).


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • Function
      Contributes to the regulation of cell cycle progression. Required for normal mitosis. Associates with the centrosome and the spindle microtubules during mitosis and functions in centrosome maturation, spindle assembly, maintenance of spindle bipolarity, centrosome separation and mitotic checkpoint control. Phosphorylates numerous target proteins, including ARHGEF2, BRCA1, KIF2A, NDEL1, PARD3, PLK1 and BORA. Regulates KIF2A tubulin depolymerase activity (By similarity). Required for normal axon formation. Plays a role in microtubule remodeling during neurite extension. Important for microtubule formation and/or stabilization.
    • Tissue specificity
      Highly expressed in testis and weakly in skeletal muscle, thymus and spleen. Also highly expressed in colon, ovarian, prostate, neuroblastoma, breast and cervical cancer cell lines.
    • Sequence similarities
      Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. Aurora subfamily.
      Contains 1 protein kinase domain.
    • Post-translational
      modifications
      Activated by phosphorylation at Thr-288; this brings about a change in the conformation of the activation segment. Phosphorylation at Thr-288 varies during the cell cycle and is highest during M phase. Autophosphorylated at Thr-288 upon TPX2 binding. Phosphorylated upon DNA damage, probably by ATM or ATR.
      Ubiquitinated by CHFR, leading to its degradation by the proteasome (By similarity). Ubiquitinated by the anaphase-promoting complex (APC), leading to its degradation by the proteasome.
    • Cellular localization
      Cytoplasm > cytoskeleton > centrosome. Cytoplasm > cytoskeleton > spindle pole. Detected at the neurite hillock in developing neurons (By similarity). Localizes on centrosomes in interphase cells and at each spindle pole in mitosis.
    • Information by UniProt
    • Database links
    • Alternative names
      • AIK antibody
      • ARK-1 antibody
      • ARK1 antibody
      • AURA antibody
      • Aurka antibody
      • Aurora 2 antibody
      • Aurora A antibody
      • Aurora kinase A antibody
      • Aurora-related kinase 1 antibody
      • Aurora/IPL1 like kinase antibody
      • Aurora/IPL1-related kinase 1 antibody
      • AURORA2 antibody
      • Breast tumor-amplified kinase antibody
      • BTAK antibody
      • hARK1 antibody
      • IAK antibody
      • IPL1 related kinase antibody
      • MGC34538 antibody
      • OTTHUMP00000031340 antibody
      • OTTHUMP00000031341 antibody
      • OTTHUMP00000031342 antibody
      • OTTHUMP00000031343 antibody
      • OTTHUMP00000031344 antibody
      • OTTHUMP00000031345 antibody
      • OTTHUMP00000166071 antibody
      • OTTHUMP00000166072 antibody
      • PPP1R47 antibody
      • Protein phosphatase 1, regulatory subunit 47 antibody
      • Serine/threonine kinase 15 antibody
      • Serine/threonine kinase 6 antibody
      • Serine/threonine-protein kinase 15 antibody
      • Serine/threonine-protein kinase 6 antibody
      • Serine/threonine-protein kinase aurora-A antibody
      • STK15 antibody
      • STK6 antibody
      • STK6_HUMAN antibody
      • STK7 antibody
      see all

    Images

    • All lanes : Anti-Aurora A antibody - Centrosome Marker (ab12875) at 1/500 dilution

      Lane 1 : Jurkat whole cell lysate
      Lane 2 : HeLa whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Alexa Fluor 680 Goat anti Rabbit IgG at 1/5000 dilution

      Performed under reducing conditions.

      Predicted band size: 46 kDa
      Observed band size: 48 kDa
      why is the actual band size different from the predicted?

    • Metaphase HeLa cells stained with ab12875 and counterstained with DAPI. The antibody shows the localisation of Aurora A to the centrosome during metaphase. 100x magnification.

      Panel 1: DNA stained with DAPI.
      Panel 2: Cells stained with ab12875 (1/500 dilution).
      Panel 3: DAPI staining is shown in red and ab12875 in green.

    • The image shows staining of formalin fixed paraffin embedded human tonsil sections with ab12875 (1/20 working dilution). Clear nuclear staining can be seen.

    References

    This product has been referenced in:
    • Eot-Houllier G  et al. Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue. Nat Commun 9:1888 (2018). Read more (PubMed: 29760389) »
    • Lee SB  et al. Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1. Mol Cell 60:21-34 (2015). IP . Read more (PubMed: 26387737) »
    See all 7 Publications for this product

    Customer reviews and Q&As

    1-10 of 10 Abreviews or Q&A

    Application
    Western blot
    Loading amount
    30 µg
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris gel)
    Sample
    Human Cell lysate - whole cell (U2OS-osteosarcoma)
    Specification
    U2OS-osteosarcoma
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

    Dr. Carlos le Sage

    Verified customer

    Submitted Jan 21 2014

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa)
    Loading amount
    20 µg
    Specification
    HeLa
    Gel Running Conditions
    Reduced Denaturing (10% SDS PAGE)
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 15°C

    Ms. AeLee Jeong

    Verified customer

    Submitted Apr 04 2013

    Answer

    Thank you for your reply.

    I apologize for the confusion, there was a difference in the numerical order of amino acids listed in the alignment I was performing. You are correct, ab115883 should not be chosen as the immunogen sequence does not overlap with the Chlamy Aurora A sequence.

    I reran the search using the proper numerical organization and came up with the following antibody:
    ab61114 (https://www.abcam.com/Aurora-A-antibody-ab61114.html): the immunogen sequence is around the human Aurora A amino acid 288. The human and Chlamy sequences are quite homologous in this region, as amino acids 285 and 288 are the only two mismatched amino acids from 275-301.

    I'm actually confident enough in the homology within this region to offer our Abtrial program for testing ab61114 in your Chlamy sequences. Information regarding this program is given below:

    To our knowledge, ab61114 has not been tested in C. reinhardtii. However by participating in our AbTrial program you can now use our products in an untested application or species without financial risk.

    Simply follow these easy steps below to apply for our AbTrial Program:

    1. Reply to this email, letting us know you are interested in testing this product.

    2. Our scientists will email you an inactive personal discount code for the value of the product.

    3. Purchase and test the product at the regular price.

    4. Submit your results, including your discount code in the additional notes section of your Abreview.

    5. Once the Abreview is submitted, the discount code will become active.

    6. Apply your discount code on your next order to receive that value off.

    The Terms and Conditions of this offer can be found at: https://www.abcam.com/abtrial

    Additionally, until 12/16/12 we are running a rabbit monoclonal antibody promotion, our "RabMab" promotion, which entitles you to a free rabbit monoclonal antibody with purchase of a primary antibody. I found a rabbit monoclonal antibody ab108353 (https://www.abcam.com/Aurora-A-antibody-EPR5026-ab108353.html) that has 54% homology with the Chlamy sequence and might be worth a try, otherwise you could choose a different Rabbit monoclonal antibody that you desire. Details of the RabMab promotion are given below:
    https://www.abcam.com/index.html?pageconfig=resource&rid=15447

    Please let me know if you have any questions about this offer and I would be happy to help you further.

    Read More

    Answer

    Thank you for contacting Abcam.

    We discussed potential anti-Aurora A antibodies to detect Chlamydomonas reinhardtii Aurora A. Since C. reinhardtii Aurora A has 67.2% homology with human Aurora A, this does not qualify for our AbTrial program, but a positive or negative Abreview can be submitted explaining your experience to give you AbPoints which can be used for an amount off of a future purchase or Amazon gift cards.

    I searched through our anti-Aurora A antibodies, and ab115883 is likely your best option should you choose to proceed
    https://www.abcam.com/aurora-a-antibody-ab115883.html

    The immunogen for this antibody is from amino acids 268-277, which has a fair amount of alignment between human and C. reinhardtii Aurora A.
    Human: http://www.uniprot.org/uniprot/O14965
    C. reinhardtii: http://www.uniprot.org/uniprot/A8ISU1

    Additionally, we currently have a RabMab promotion running until 12/16/12, which entitles you to a Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last. Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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

    Read More

    Answer

    Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

    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 of ab13824. This is shipping to the same address as your original order.

    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.

    Read More

    Question

    To prepare my sample, I used a buffer containing 50mM Tris pH 7.5, 150mM NaCl, 1mM MgCl2 and 1% triton X-100. I included proteases inhibitors in the lysis buffer (complete edta-free tablet, from Roche). I incubated the cells 20 minutes with
    this buffer and then cleared the lysate by a 15 min centrifugation at 20 000g. I evaluated the protein concentration of my
    samples by the bradford method and then I added SDS-sample loading buffer to the lysates (containing DTT). I boiled the
    samples for 5 minutes.
    I suppose that the 8% acrylamide gel I used for SDS-PAGE is suitable for the experiment I performed since I see the 31kDa
    band of the marker, meaning that proteins around 48 kDA might still be in the gel, although I could also try with a 12%
    acrylamide gel.
    There was no SDS in my transfer buffer, only methanol, tris and glycine. I did a Ponceau coloration of the membrane to
    make sure the transfer worked well and everything was normal. I can also precise that I used these samples for other gels
    that were runned and transferred at the same time as the gel described for the western blot that did not worked with
    ab12875. I was able to detect 3 other proteins by Western blot, the only experiment that did not work was the one using
    ab12875 primary antibody. Those proteins had a size of 60, 55 and 42 kDa so there is no reason why I should not see Aurora A which is supposed to migrate at 48 kDa.
    We purchased this antibody less than one month ago so it might be possible that we request a credit. According to the catalog, Abcam sells other anti Aurora A antibodies that were reported to work well in western blot experiments so we would
    be interested in trying another one.

    Read More
    Answer

    Thank you for your reply.

    I would be happy to offer you a replacement antibody or a credit. Please let me know which you would prefer. If you would like an antibody, please include the catalogue number.

    I look forward to your reply.

    Read More

    Question
    Answer

    Thank you for taking time to contact us. 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.

    Can you tell me a bit more about how you prepared your cell lysate? What kind of lysis buffer did you use? We would recommend NP-40 or RIPA buffer.

    Are you including protease inhibitors in with the sample preparation? I see that the samples are denatured, but are they reduced as well with b-mercaptoethanol or DTT? Did you boil the samples for 10 mins?

    Do you have access to a higher percentage gel? You may want to try a 12% gel for a protein below 50 kDa.

    For transfer of small proteins (< 100 kDa), consider removing SDS from the transfer buffer and keep the methanol concentration at 20%.

    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
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cultured Cells (Head and neck cancer cell)
    Specification
    Head and neck cancer cell
    Fixative
    Acetone
    Permeabilization
    No
    Blocking step
    FBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 27°C

    Abcam user community

    Verified customer

    Submitted Jan 04 2010

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Bladder)
    Specification
    Bladder
    Fixative
    Paraformaldehyde
    Antigen retrieval step
    Heat mediated
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10%

    Abcam user community

    Verified customer

    Submitted Jan 23 2007

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry
    Sample
    Human Cell (HeLa and SK-N-SH)
    Specification
    HeLa and SK-N-SH
    Fixative
    Paraformaldehyde

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Oct 12 2005

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

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