Overview

  • Product name
    Anti-CDKN2A/p14ARF antibody [ARF 4C6/4]
    See all CDKN2A/p14ARF primary antibodies
  • Description
    Mouse monoclonal [ARF 4C6/4] to CDKN2A/p14ARF
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ELISA, WB, ICC/IF, IPmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    His-tagged p14ARF protein expressed in bacteria.

Properties

Applications

Our Abpromise guarantee covers the use of ab11048 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 Use 0.5µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

ELISA Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 14 kDa.
ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

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

  • ICC/IF image of ab11048 stained human MCF7 cells. The cells were 4% PFA 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 (ab11048, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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). This antibody also gave a positive IF result in HeLa, Hek293 and HepG2cells.
  • Overlay histogram showing HeLa cells stained with ab11048 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab11048, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
  • Lee CK  et al. Syk-mediated tyrosine phosphorylation of mule promotes TNF-induced JNK activation and cell death. Oncogene 35:1988-95 (2016). Read more (PubMed: 26212014) »
  • Hamilton G  et al. AKT regulates NPM dependent ARF localization and p53mut stability in tumors. Oncotarget 5:6142-67 (2014). Read more (PubMed: 25071014) »
See all 5 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for contacting us.

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below;

https://www.abcam.com/ps/pdf/protocols/Sandwich%20ELISA.pdf

https://www.abcam.com/ps/pdf/protocols/Direct%20ELISA%20protocol.pdf

https://www.abcam.com/ps/pdf/protocols/Direct%20ELISA%20protocol.pdf

https://www.abcam.com/ps/pdf/protocols/Indirect%20ELISA%20protocol.pdf

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

Dear technical support team: This customer has purchased ab11048 (Anti-CDKN2A/p14ARF antibody [ARF 4C6/4]), and he raised an enquiry about his ICC data which was staining in cell nucleus. However, according to the online reference indicated this protein should expression in cell nucleolar. Therefore, this customer would like to ask for your help to offer some suggestion to him. I also attached his ICC data in this letter, and his experiment step as follow:   1) Order details: ˙Batch number ( Lot number): GR37995-1 ˙Abcam product code: ab11048 ˙Antibody storage conditions (temperature/reconstitution etc) :-20℃ 2) Please describe the problem (high background, non-specific signal…etc). The signal should in nucleolar 3) On what material are you testing the antibody in IHC/ICC? ˙Species: human ˙What’s cell line or tissue: U2OS cell 4) Sample preparation: ˙Type of sample (Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed ˙paraffin embedded sections, cells in culture, other:____) : cell culture ˙Sample preparation: cells seed in coverslip 5) Fixation step ˙Yes or No : yes ˙If yes, Fixative agent and concentration: 4% paraformaldehyde ˙Fixation time: 10min ˙Fixation temperature : room templature 6) Antigen retrieval method(including time, temperature etc.): no   7) Permeabilization method: ˙Permeabilizing agent and concentration: 0.5% Triton X-100 in PBS 8) Blocking agent (eg BSA, serum…): no blocking 9) ˙Endogenous peroxidases blocked? no ˙Endogenous biotins blocked? no 10) Primary antibody (If more than one was used, describe in “additional notes”) : ˙Species: mouse monoclonal ˙Reacts against: p14ARF ˙At what dilution(s) have you tested this antibody: 1:25 ˙Diluent buffer:  PBS ˙Incubation time: overnight ˙Incubation temperature: 4℃ ˙What washing steps were done (which buffer, number of washes): PBS wash three times 11)  Secondary antibody: ˙Species:goat ˙Reacts against which species: mouse ˙At what dilution(s) have you tested this antibody: 1:100 ˙Diluent buffer:PBS ˙Incubation time:1 hour in RT ˙What washing steps were done (which buffer, number of washes): PBS wash three times ˙Fluorochrome or enzyme conjugate (eg: FITC, HRP, AP, biotin…etc):FITC ˙Do you know whether the problems you are experiencing come from the secondary? The secondary antibody is working in other experiment 12) Signal amplification method (eg: ABC, LSAB, HRP polymer, TSE): no 13) Detection method (eg: DAB, BCIP/NBT …etc): no 14) ˙ How many times have you run this staining? Three times ˙Do you obtain the same results every time?  Yes ˙What steps have you altered to try and optimize the use of this antibody? 1. Fixation buffer(paraformaldehyde, formaldehyde or acetone) 2. incubation condition of first antibody   Could you please help this customer to solve the problem? Thanks for your kindly help Best regards

Read More
Answer

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product. According to Swiss-Prot, the localization of the staining (CDKN2A/p14ARF) could be both cytoplasmic and nuclear. You may wish to take a look at this site for further information: http://www.uniprot.org/uniprot/P42771 U2OS is a human osteosarcoma cell line; it is known that CDKN2A/p14ARF is widely expressed but not detected in certain tissue such brain or skeletal muscle. There are at least 4 different splice variants and the expression pattern of these isoforms (P42771-1, P42771-2, P42771-3, Q8N726-1) may vary in different tissue/cell types. After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions: 1) Try to use cells which are intensively dividing and the cell culture should in exponential (log) phase of growth. 2) It may be worth staining HeLa cells or sections of human liver or lymphomas as good positive control. 3) In order to increase the signal, it would be worth applying ABC signal enhancing system. I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More

Question
Answer

Thank you for your enquiry. Our source for this antibody has told me that ab11048 does detect endogenous p14ARF; please see the following publication for additional information: Llanos S et al. Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat Cell Biol 3:445-52 (2001). PubMed: 11331871 If you have any additional questions, please contact us again.

Read More

Question

Thanks a lot. I did a PubMed search for "HeLa cells and ARF", and found a paper, which shows the band detected by the same antibody as that I obtained from your company. I am looking for some HeLa cells, hopefully to get some positive control samples. Have a nice weekend. Oncogene. 2002 Oct 3;21(44):6779-90. Human p14(Arf): an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function. David-Pfeuty T, Nouvian-Dooghe Y. UMR 146 du CNRS, Institut Curie-Recherche, Batiment 110, Centre Universitaire, 91405 Orsay Cedex, France. Therese.Pfeuty@curie.u-psud.fr The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16(Ink4a) which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14(Arf), a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14(Arf) and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14(Arf) predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon short-term treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14(Arf) might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.

Read More
Answer

Thank you for the information about the paper, if you have any more questions, just let us know.

Read More

Question
Answer

Thank you for your enquiry. Although I was unable to find out exactly what the originator used as a positive control, they have said that Hela cells should be appropriate to use as a positive control for this antibody. If you have any more questions, please contact me again.

Read More

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

Sign up