Validated using a knockout cell line
Recombinant
RabMAb

Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620)

Overview

  • Product name
    Anti-CDKN2A/p14ARF antibody [EPR17878]
    See all CDKN2A/p14ARF primary antibodies
  • Description
    Rabbit monoclonal [EPR17878] to CDKN2A/p14ARF
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IP, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human CDKN2A/p14ARF aa 50 to the C-terminus. The exact sequence is proprietary.
    Database link: Q8N726

  • Positive control
    • WB: HeLa whole cell lysate transfected with CDKN2A/p14ARF with His-tag; PC-3 and HEK-293 whole cell lysates. ICC/IF: HeLa cells transfected with CDKN2A/p14ARF. Flow Cyt: HeLa cells transfected with CDKN2A/p14ARF-GFP. IP: CDKN2A/p14ARF transfected HeLa whole cell lysate.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab185620 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
Flow Cyt 1/120.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/250.
IP 1/80.
WB 1/1000. Detects a band of approximately 14 kDa (predicted molecular weight: 14 kDa).

Target

  • Relevance
    The gene for CDK2NA generates several transcripts/proteins which differ from each other in their first exons. Three of these transcripts are generated by alternative splicing (isoform 1 a.k.a p16INK4A, isoform 2 and isoform 3 a.k.a p12), two of which are known to function as inhibitors of CDK4 kinase. One other transcript that is generated from this gene contains an alternate reading frame (ARF), with the first exon located 20kb upstream of the remainder of the gene(isoform 4 a.k.a. p14ARF, p19ARF, ARF). In spite of the structural and some functional differences, all the proteins encoded by the CDKN2A gene are involved in cell cycle G1 control.
  • Cellular localization
    Cytoplasmic and Nuclear
  • Database links
  • Alternative names
    • ARF antibody
    • CDK4 inhibitor p16 INK4 antibody
    • CDK4I antibody
    • CDKN2 antibody
    • CDKN2A antibody
    • Cell cycle negative regulator beta antibody
    • CMM2 antibody
    • Cyclin dependent kinase 4 inhibitor A antibody
    • Cyclin dependent kinase inhibitor 2A antibody
    • INK4 antibody
    • INK4a antibody
    • Melanoma p16 inhibits CDK4 antibody
    • MLM antibody
    • MTS 1 antibody
    • MTS1 antibody
    • Multiple tumor suppressor 1 antibody
    • p14 antibody
    • p16 antibody
    • p16 INK4a antibody
    • p16INK4a antibody
    • p19 antibody
    • P19ARF antibody
    • TP16 antibody
    see all

Images

  • All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/1000 dilution

    Lane 1 : Wild-type HeLa whole cell lysate
    Lane 2 : CDKN2A knockout HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 14 kDa



    Lanes 1 - 2: Merged signal (red and green). Green - ab185620 observed at 14 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab185620 was shown to specifically react with CDKN2A/p14ARF in wild-type HeLa cells as signal was lost in CDKN2A knockout cells. Wild-type and CDKN2A knockout samples were subjected to SDS-PAGE. Ab185620 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/5000 dilution

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate transfected with empty vector (vector control)
    Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate transfected with CDKN2A/p14ARF with His-tag

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 14 kDa


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-CDKN2A/p14ARF antibody [EPR17878] (ab185620) at 1/1000 dilution

    Lane 1 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
    Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 14 kDa
    Observed band size: 14 kDa


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The expression level of p14 ARF is relatively low in most cell types and will be induced by oncogene expression. (PMID: 9694807; PMID: 9694806).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma) cells transfected with CDKN2A/p14ARF or empty vector, labeling CDKN2A/p14ARF with ab185620 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution 

    The nuclear counterstain is DAPI (blue).

    Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

    The negative controls are as follows:-

    -ve control 1: ab185620 at 1/250 dilution followed by ab150120 at 1/1000 dilution.

    -ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells transfected with CDKN2A/p14ARF-GFP labeling CDKN2A/p14ARF with ab185620 at 1/120 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti Rabbit IgG (PE) at 1/150 dilution was used as the secondary antibody.

  • CDKN2A/p14ARF was immunoprecipitated from 1mg of CDKN2A/p14ARF transfected HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab185620 at 1/80 dilution.

    Western blot was performed from the immunoprecipitate using ab185620 at 1/1000 dilution.

    VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/10000 dilution.

    Lane 1: CDKN2A/p14ARF transfected HeLa whole cell lysate, 10µg (Input).

    Lane 2: ab185620 IP in CDKN2A/p14ARF transfected HeLa whole cell lysate.

    Lane 3: Rabbit IgG,monoclonal[EPR25A] - Isotype Control (ab172730) instead of ab185620 in CDKN2A/p14ARF transfected HeLa whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 1 second.

References

This product has been referenced in:
  • Coleman KE  et al. SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function. Elife 6:N/A (2017). Read more (PubMed: 28475037) »

See 1 Publication for this product

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

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