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    cenpf-antibody-ab5.pdf

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Epigenetics and Nuclear Signaling Chromosome Structure Centromeres
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Anti-CENPF antibody (ab5)

  • Datasheet
  • SDS
Reviews (7)Q&A (2)References (70)

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Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
  • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPF antibody (ab5)
  • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
  • Western blot - Anti-CENPF antibody (ab5)
  • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)

Key features and details

  • Rabbit polyclonal to CENPF
  • Suitable for: ICC/IF, IHC-P, WB
  • Reacts with: Human
  • Isotype: IgG

Get better batch-to-batch reproducibility with a recombinant antibody

Product image
Anti-CENPF antibody [EPR20406] (ab223847)
  • Research with confidence – consistent and reproducible results with every batch
  • Long-term and scalable supply – powered by recombinant technology for fast production
  • Success from the first experiment – confirmed specificity through extensive validation
  • Ethical standards compliant – production is animal-free

Overview

  • Product name

    Anti-CENPF antibody
    See all CENPF primary antibodies
  • Description

    Rabbit polyclonal to CENPF
  • Host species

    Rabbit
  • Tested Applications & Species

    Application Species
    ICC/IF
    Human
    IHC-P
    Human
    WB
    Human
    See all applications and species data
  • Immunogen

    Fusion protein with C-terminus of CENP-F (Human).

  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.4
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 50% Glycerol (glycerin, glycerine)
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Chromosome Structure
    • Centromeres
    • Epigenetics and Nuclear Signaling
    • Cell cycle
    • Chromosome Structure
    • Centromere

Associated products

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab5 in the following tested applications.

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

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
ICC/IF
Human
IHC-P
Human
WB
Human
Application Abreviews Notes
ICC/IF (5)
1/400 - 1/750.
IHC-P
Use at an assay dependent concentration.
WB
1/1500. Detects a band of approximately 330 kDa (predicted molecular weight: 330 kDa). Block membranes for 1 hour with 5% nonfat dry milk/TBS-T.
Notes
ICC/IF
1/400 - 1/750.
IHC-P
Use at an assay dependent concentration.
WB
1/1500. Detects a band of approximately 330 kDa (predicted molecular weight: 330 kDa). Block membranes for 1 hour with 5% nonfat dry milk/TBS-T.

Target

  • Function

    Required for kinetochore function and chromosome segregation in mitosis. Required for kinetochore localization of dynein, LIS1, NDE1 and NDEL1. Regulates recycling of the plasma membrane by acting as a link between recycling vesicles and the microtubule network though its association with STX4 and SNAP25. Acts as a potential inhibitor of pocket protein-mediated cellular processes during development by regulating the activity of RB proteins during cell division and proliferation. May play a regulatory or permissive role in the normal embryonic cardiomyocyte cell cycle and in promoting continued mitosis in transformed, abnormally dividing neonatal cardiomyocytes. Interaction with RB directs embryonic stem cells toward a cardiac lineage. Involved in the regulation of DNA synthesis and hence cell cycle progression, via its C-terminus. Has a potential role regulating skeletal myogenesis and in cell differentiation in embryogenesis. Involved in dendritic cell regulation of T-cell immunity against chlamydia.
  • Involvement in disease

    Stromme syndrome
  • Sequence similarities

    Belongs to the centromere protein F family.
  • Developmental stage

    Gradually accumulates during the cell cycle, reaching peak levels in G2 and M phase, and is rapidly degraded upon completion of mitosis.
  • Post-translational
    modifications

    Hyperphosphorylated during mitosis.
  • Cellular localization

    Cytoplasm, perinuclear region. Nucleus matrix. Chromosome, centromere, kinetochore. Cytoplasm, cytoskeleton, spindle. Relocalizes to the kinetochore/centromere (coronal surface of the outer plate) and the spindle during mitosis. Observed in nucleus during interphase but not in the nucleolus. At metaphase becomes localized to areas including kinetochore and mitotic apparatus as well as cytoplasm. By telophase, is concentrated within the intracellular bridge at either side of the mid-body.
  • Target information above from: UniProt accession P49454 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 1063 Human
    • Omim: 600236 Human
    • SwissProt: P49454 Human
    • Unigene: 497741 Human
    • Alternative names

      • AH antigen antibody
      • Cell cycle dependent 350K nuclear protein antibody
      • CENF antibody
      • CENP F antibody
      • CENP F kinetochore protein antibody
      • CENP-F antibody
      • CENPF antibody
      • CENPF kinetochore protein antibody
      • CENPF_HUMAN antibody
      • Centromere protein F 350/400ka antibody
      • Centromere protein F antibody
      • Centromere protein F, 350/400kDa antibody
      • CILD31 antibody
      • Hcp 1 antibody
      • Hcp1 antibody
      • Kinetochore protein CENP F antibody
      • Kinetochore protein CENPF antibody
      • Mitosin antibody
      • PRO1779 antibody
      • STROMS antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)This image is courtesy of Kirk McManus, University of British Columbia

      HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image demonstrates the dramatic increase in fluorescence that occurs late in G2 cells (indicated by arrows). In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are coloured green and red respectively. 40x magnification.

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)

      ICC/IF image of ab5 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab5, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPF antibody (ab5)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPF antibody (ab5)
      Ab5 staining Human normal colon tissue. Staining is localised to nuclear compartment.
      Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
      Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
    • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)This image is courtesy of Kirk McManus, University of British Columbia

      Interpahse and Prophase HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image emphasizes the redistribution of CENPF from the nuclear matrix during late G2 following entry into the initial stages of mitosis (see the accompanying image). Distinct punctate CENPF patterns proximally located in relation to the centromeres can be seen. In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are green and red respectively. 100x magnification. 

    • Western blot - Anti-CENPF antibody (ab5)
      Western blot - Anti-CENPF antibody (ab5)
      All lanes : Anti-CENPF antibody (ab5) at 1/1500 dilution

      Lane 1 : Mitotic HeLa Lysate
      Lane 2 : Asynchronous HeLa Lysate
      Lane 3 : 50gamma Mitotic HeLa Lysate
      Lane 4 : 50gamma Asynchronous HeLa Lysate

      Lysates/proteins at 25 µg per lane.

      Predicted band size: 330 kDa
      Observed band size: 310 kDa why is the actual band size different from the predicted?

    • Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)
      Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (ab5)This image is courtesy of an Abreview submitted by Dr. Stuart Rulten.

      Paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells stained for CENPF (red) using ab5 at 1/500 dilution in ICC/IF. ). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) at 1/500 dilution was used as the secondary antibody.

      See Abreview

    Protocols

    • Recommended WB protocol for ab5

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (70)

    Publishing research using ab5? Please let us know so that we can cite the reference in this datasheet.

    ab5 has been referenced in 70 publications.

    • Auckland P  et al. CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes. J Cell Biol 219:N/A (2020). PubMed: 32207772
    • Stiff T  et al. Prophase-Specific Perinuclear Actin Coordinates Centrosome Separation and Positioning to Ensure Accurate Chromosome Segregation. Cell Rep 31:107681 (2020). PubMed: 32460023
    • Shen T  et al. XBP1 negatively regulates CENPF expression via recruiting ATF6a to the promoter during ER stress. Cancer Cell Int 20:459 (2020). PubMed: 32973403
    • Dwivedi D  et al. The dynein adaptor Hook2 plays essential roles in mitotic progression and cytokinesis. J Cell Biol 218:871-894 (2019). PubMed: 30674580
    • Martinez-Macias MI  et al. FUS (fused in sarcoma) is a component of the cellular response to topoisomerase I-induced DNA breakage and transcriptional stress. Life Sci Alliance 2:N/A (2019). PubMed: 30808650
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-9 of 9 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPF antibody

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 23°C
    Sample
    Mouse Cell (Mouse embryonic fibroblast cells)
    Specification
    Mouse embryonic fibroblast cells
    Permeabilization
    Yes - 0.3% Triton X-100
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Oct 15 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPF antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Human lung adenocarcinoma A549)
    Specification
    Human lung adenocarcinoma A549
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.2% Triton in PBS
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Dr. Stuart Rulten

    Verified customer

    Submitted Jan 02 2013

    Question

    I am looking for a positive control for use in WB and I notice that your image shows a strong band in mitotic cells. Can you tell me how these cells were prepared to ensure that they are all mitotic?

    Read More

    Abcam community

    Verified customer

    Asked on Jul 11 2012

    Answer

    Thank you for contacting us.

    While I was unable to locate any information regarding the specific protocol used to synchronize the mitotic cells in the image on our website (which was provided by a collaborator), the following protocol may be helpful.

    Thymidine-Nocodazole Block:

    HeLa cells were plated at a density of 2x106in 150 mm tissue culture dishes in DMEM with 10%FBS and 100U Pen-Strep
    24 hours after plating, cells were arrested with2mM Thymidine for17-18 hours(S-phase block)
    After Thymidine block: remove Thymidine by washing with 1xPBS
    Add fresh DMEM with 10%FBS and 100U Pen-Strep for 4 hours to release cells.
    Add 100ng/ml Nocodazole to the Media for 12 hours (mitotic block)
    Floating mitotic cells were collected, washed twice in 1X PBS, and replated at a density of 4x106 cells in each 150 cm dish.



    This protocol was used to produce the cells in Figure 1 B of this paper: http://www.molbiolcell.org/content/13/6/1977.long

    I hope this helps, please let me know if you need any additional information or assistance.

    Read More

    Abcam Scientific Support

    Answered on Jul 11 2012

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPF antibody

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa cells)
    Specification
    HeLa cells
    Fixative
    Formaldehyde
    Permeabilization
    Yes - 0.1% Triton X 100
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 29 2008

    Flow Cytometry abreview for Anti-CENPF antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Flow Cytometry
    Sample
    Human Cell (T98G glioma cells)
    Specification
    T98G glioma cells
    Fixation
    Ethanol
    Permeabilization
    Yes - Ethanol
    Gating Strategy
    intact cells with >80% and <250% of G1 DNA content
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 29 2008

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPF antibody

    Good
    Abreviews
    Abreviews
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Paraformaldehyde
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 15 2006

    Question

    I am interested in using this antibody for immunohistochemistry on mouse tissue, as well as attempting chromatin precipitation assays. The datasheet doesn't specify exactly which region of the human protein was used to make the antibody. Can you inform me of the peptide sequence used to raise the antibody so I can identify the level of conservation in the mouse and assess its likelihood of binding to the mouse protein.

    Read More

    Abcam community

    Verified customer

    Asked on Jan 24 2006

    Answer

    The exact immunogen is proprietary information which was not provided by the supplier unfortunately. I BLASTed the last 200+ residues (our datasheet says that it was made from a bacterial fusion protein from the C-terminus of CENP-F). It shows ~75% homology with fragmented mouse protein. Granted, this is a large span of residues to compare but this is a large protein and this is my best guess for where the epitope resides. I hope that this will already help you and am sorry I couldn't help you more,

    Read More

    Abcam Scientific Support

    Answered on Jan 30 2006

    Immunocytochemistry abreview for Anti-CENPF antibody

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry
    Sample
    Human Cell (HeLa, T98G)
    Specification
    HeLa, T98G
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1%
    Read More

    Dr. Lesley McPhail

    Verified customer

    Submitted Nov 09 2005

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CENPF antibody

    Excellent
    Abreviews
    Abreviews
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa, SK-N-SH and HCT116)
    Specification
    HeLa, SK-N-SH and HCT116
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 13 2005

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