Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human, Mouse

Cited:

Human, Mouse

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Knockdown Validated

Cited:

Western Blot, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all p14ARF/CDKN2A Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to a portion of human p14ARF (between residues 50-132). [Swiss-Prot# Q8N726]

Reactivity Notes

Human and mouse reactivity reported in scientific literature.

Localization

Nuclear

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Description

Novus Biologicals Rabbit p14ARF/CDKN2A Antibody - BSA Free (NB200-111) is a polyclonal antibody validated for use in IHC, WB, Flow, ICC/IF and IP. Anti-p14ARF/CDKN2A Antibody: Cited in 18 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.

Scientific Data Images for p14ARF/CDKN2A Antibody - BSA Free

Knockdown Validated: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

p14ARF-CDKN2A-Antibody-Western-Blot-NB200-111-img0012.jpg
Immunohistochemistry: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Immunohistochemistry: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

p14ARF-CDKN2A-Antibody-Immunocytochemistry-Immunofluorescence-NB200-111-img0011.jpg
Immunocytochemistry/ Immunofluorescence: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Immunocytochemistry/ Immunofluorescence: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Immunocytochemistry/Immunofluorescence: p14ARF/CDKN2A Antibody [NB200-111] - p14ARF antibody was tested in HeLa cells with Dylight 488 (green). Nuclei were counterstained with DAPI (blue). Tubulin was stained with alpha tubulin (red).
Western Blot: p14ARF/CDKN2A AntibodyBSA Free [NB200-111]

Western Blot: p14ARF/CDKN2A AntibodyBSA Free [NB200-111]

Western Blot: p14ARF/CDKN2A Antibody [NB200-111] - ARF expression levels in OCI/AML3 cells after proteasome treatment. This image was submitted via customer Review.
Flow Cytometry: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Flow Cytometry: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Flow Cytometry: p14ARF/CDKN2A Antibody [NB200-111] - p14ARF antibody was tested at 1:400 in HeLa cells using an Alexa Fluor 488 secondary (shown in purple). M1 is defined by unstained cells.
Western Blot: p14ARF/CDKN2A AntibodyBSA Free [NB200-111]

Western Blot: p14ARF/CDKN2A AntibodyBSA Free [NB200-111]

Western Blot: p14ARF/CDKN2A Antibody [NB200-111] - Analysis of HeLa Whole Cell Lysate (NB800-PC1) using p14ARF antibody (lot C) employing ECL detection method.
Immunohistochemistry-Frozen: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Immunohistochemistry-Frozen: p14ARF/CDKN2A Antibody - BSA Free [NB200-111]

Immunohistochemistry-Frozen: p14ARF/CDKN2A Antibody [NB200-111] - Mouse brain section, hippocampal (10 um thickness). 3% PFA perfused. 1:1000 dilultion. Image from verified customer review. Image using the Biotin form of this antibody.
p14ARF/CDKN2A Antibody - BSA Free

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT decreases p53mut stability(A) T24 cells & PSN1 cells were treated with MK-2206 (5 μM) or DMSO for 24 hrs. Cells were fixed & stained with DAPI & anti-mutant p53 (OP29 clone). (B) T24 cells were pre-treated with MK-2206 (5 μM) or DMSO for 24 hrs before the addition of fresh media containing cyclohexamide (100 μM) (CHX) in combination with MK-2206 (5 μM) or DMSO for the times indicated. Nuclear extracts were prepared from treated cells & blotted with the indicated antibodies. Quantification is relative to initiation of CHX treatment for both conditions *, MK-2206 is 40% of DMSO control but taken as 1.0 for relative assessment. (C) H1299 cells were transfected with HA-tagged-ubiquitin & mutant p53 (R175H or R248W) as indicated. Transfected cells were treated with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) for 16hrs as indicated. p53 immunoprecipitates & whole cell lysates were probed with the indicated antibodies. (D) T24 cells were transfected with non-targeting control or p14ARF siRNA & treated with DMSO, MK-2206 (5 μM) or the NPM oligomerisation inhibitor NCS348884 (4 μM) (Qi et al., 2008) as indicated. (E) T24 cells were transfected with non-targeting control, AKT1, or p14ARF siRNA. Cells were treated with NCS348884 (4 μM), Nutlin3A (5 μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. (F) KPC mice derived KRASG12D p53 Floxed (p53Fl), KRASG12D p53R172H ARF+/+ & KRASG12D p53R172H ARF−/− pancreatic tumor cells were treated with MK-2206 (1μM), Nutlin3A (5μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
p14ARF/CDKN2A Antibody - BSA Free

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT decreases p53mut stability(A) T24 cells & PSN1 cells were treated with MK-2206 (5 μM) or DMSO for 24 hrs. Cells were fixed & stained with DAPI & anti-mutant p53 (OP29 clone). (B) T24 cells were pre-treated with MK-2206 (5 μM) or DMSO for 24 hrs before the addition of fresh media containing cyclohexamide (100 μM) (CHX) in combination with MK-2206 (5 μM) or DMSO for the times indicated. Nuclear extracts were prepared from treated cells & blotted with the indicated antibodies. Quantification is relative to initiation of CHX treatment for both conditions *, MK-2206 is 40% of DMSO control but taken as 1.0 for relative assessment. (C) H1299 cells were transfected with HA-tagged-ubiquitin & mutant p53 (R175H or R248W) as indicated. Transfected cells were treated with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) for 16hrs as indicated. p53 immunoprecipitates & whole cell lysates were probed with the indicated antibodies. (D) T24 cells were transfected with non-targeting control or p14ARF siRNA & treated with DMSO, MK-2206 (5 μM) or the NPM oligomerisation inhibitor NCS348884 (4 μM) (Qi et al., 2008) as indicated. (E) T24 cells were transfected with non-targeting control, AKT1, or p14ARF siRNA. Cells were treated with NCS348884 (4 μM), Nutlin3A (5 μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. (F) KPC mice derived KRASG12D p53 Floxed (p53Fl), KRASG12D p53R172H ARF+/+ & KRASG12D p53R172H ARF−/− pancreatic tumor cells were treated with MK-2206 (1μM), Nutlin3A (5μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
p14ARF/CDKN2A Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Immunocytochemistry/ Immunofluorescence: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT modulates p53 stability in-vivo & synergizes with ionizing radiation to inhibit tumor growth(A) PSN1 xenografts (PSN1 cells co-injected with LTC-14 stellate cells) established in the flank of athymic nude mice were treated with MK-2206 (60 mg/kg-320 mg/kg) as indicated or beta -cyclo-dextrin (1.5 mg/ml) carrier. Xenograft tumors were lysed & lysates probed by western blot with the indicated antibodies. (B-D) Sections of PSN1 xenografts treated with three consecutive doses of MK-2206 (60 mg/kg). (B) Sections of PSN1 xenografts & in-vitro PSN1 cells fixed & stained with anti-NPM (red) & anti-p14ARF (green). (C) PSN1 xenografts treated with MK-2206 or carrier were stained with DAPI, anti-p53 (DO1) or p53mut (OP29 clone) (D) PSN1 xenografts treated with MK-2206 (60 mg/kg) or carrier were stained by immunohistochemical methods with anti-pS473-AKT, anti-phospho-S48-NPM (pS48-NPM) or p53. (E) PSN1 xenografts established in the flank of athymic nude mice were injected subcutaneously with two alternate day doses of MK-2206 (60 mg/kg) or carrier. Mice were subsequently treated with a single dose of IR (6 Gy) & tumor volumes measured regularly with callipers. Dash lines indicate tumor growth differential at 250 mm3. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
p14ARF/CDKN2A Antibody - BSA Free

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT decreases p53mut stability(A) T24 cells & PSN1 cells were treated with MK-2206 (5 μM) or DMSO for 24 hrs. Cells were fixed & stained with DAPI & anti-mutant p53 (OP29 clone). (B) T24 cells were pre-treated with MK-2206 (5 μM) or DMSO for 24 hrs before the addition of fresh media containing cyclohexamide (100 μM) (CHX) in combination with MK-2206 (5 μM) or DMSO for the times indicated. Nuclear extracts were prepared from treated cells & blotted with the indicated antibodies. Quantification is relative to initiation of CHX treatment for both conditions *, MK-2206 is 40% of DMSO control but taken as 1.0 for relative assessment. (C) H1299 cells were transfected with HA-tagged-ubiquitin & mutant p53 (R175H or R248W) as indicated. Transfected cells were treated with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) for 16hrs as indicated. p53 immunoprecipitates & whole cell lysates were probed with the indicated antibodies. (D) T24 cells were transfected with non-targeting control or p14ARF siRNA & treated with DMSO, MK-2206 (5 μM) or the NPM oligomerisation inhibitor NCS348884 (4 μM) (Qi et al., 2008) as indicated. (E) T24 cells were transfected with non-targeting control, AKT1, or p14ARF siRNA. Cells were treated with NCS348884 (4 μM), Nutlin3A (5 μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. (F) KPC mice derived KRASG12D p53 Floxed (p53Fl), KRASG12D p53R172H ARF+/+ & KRASG12D p53R172H ARF−/− pancreatic tumor cells were treated with MK-2206 (1μM), Nutlin3A (5μM) or DMSO as indicated. Whole cell lysates were probed with the indicated antibodies. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
p14ARF/CDKN2A Antibody - BSA Free

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT promotes enhanced MDM2 activity via the increased association between NPM & p14ARF(A) Npm−/−, p53−/−double null MEF were infected with pBABE retrovirus empty vector & pBABE expressing FLAG-tagged-NPM-WT, NPM-S48A or S48E as indicated. Immunopurification of NPM was done by pulling down with the Flag tag (middle panel) followed by elution of complexes by the Flag peptide & subsequent immunopurification of endogenous MDM2 (lower panel). (B) Nuclear immunoprecipitates of MDM2 from T24 cells treated with MK-2206 (5 μM, 24 hrs). Immunoprecipitates & lysates were blotted with the indicated antibodies. (C) T24 cells were treated with MK-2206 (5 μM) as indicated. p14ARF was immunoprecipitated from whole cell lysates & nuclear extracts & the association with NPM & MDM2 determined by western blot. Immunoprecipitates & lysates were blotted with the indicated antibodies. (D) MDM2 & (E) p53 ubiquinitation assay in H1299 cells transfected with wild type p53, HA-tagged ubiquitin & treated for 16 hrs with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) as indicated. Immunoprecipitates & whole cell lysates were probed with the indicated antibodies. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
p14ARF/CDKN2A Antibody - BSA Free

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] -

Western Blot: p14ARF/CDKN2A Antibody - BSA Free [NB200-111] - Inhibition of AKT promotes enhanced MDM2 activity via the increased association between NPM & p14ARF(A) Npm−/−, p53−/−double null MEF were infected with pBABE retrovirus empty vector & pBABE expressing FLAG-tagged-NPM-WT, NPM-S48A or S48E as indicated. Immunopurification of NPM was done by pulling down with the Flag tag (middle panel) followed by elution of complexes by the Flag peptide & subsequent immunopurification of endogenous MDM2 (lower panel). (B) Nuclear immunoprecipitates of MDM2 from T24 cells treated with MK-2206 (5 μM, 24 hrs). Immunoprecipitates & lysates were blotted with the indicated antibodies. (C) T24 cells were treated with MK-2206 (5 μM) as indicated. p14ARF was immunoprecipitated from whole cell lysates & nuclear extracts & the association with NPM & MDM2 determined by western blot. Immunoprecipitates & lysates were blotted with the indicated antibodies. (D) MDM2 & (E) p53 ubiquinitation assay in H1299 cells transfected with wild type p53, HA-tagged ubiquitin & treated for 16 hrs with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) as indicated. Immunoprecipitates & whole cell lysates were probed with the indicated antibodies. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25071014), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for p14ARF/CDKN2A Antibody - BSA Free

Application
Recommended Usage

Flow Cytometry

1:400

Immunocytochemistry/ Immunofluorescence

reported in scientific literature (PMID 25071014)

Immunohistochemistry

1:10-1:500. Use reported in scientific literature (PMID 18505964)

Immunohistochemistry-Frozen

1:10-1:500. Use reported by customer review

Immunohistochemistry-Paraffin

1:10-1:500

Immunoprecipitation

reported in scientific literature (PMID 20699639)

Western Blot

reported in scientific literature (PMID 21636682)
Application Notes
This p14ARF antibody can be used for Western blotting, where a band is seen at ~16 kDa, representing p14ARF. Additional faint bands may be seen at ~32 and 47 kDa. In ICC/IF [PMID: 25071014], nuclear focal staining was observed in HeLa cells.

Reviewed Applications

Read 2 reviews rated 4 using NB200-111 in the following applications:

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.05% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: p14ARF/CDKN2A

The INK4a-ARF locus is comprised of two tumor suppressors, p16INK4a and p14ARF. These two proteins are encoded through differential splicing of alternative first exons. The p16INK4a (exon 1 alpha) protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product complexes with the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. P14ARF is a potent tumor suppressor in the presence of wild-type p53, while mutant p53 substantially reduces growth inhibition by p14ARF.

Alternate Names

ARF, CDK4 inhibitor p16-INK4, CDK4IP14ARF, CDKN2, cell cycle negative regulator beta, CMM2P16-INK4A, Cyclin-dependent kinase 4 inhibitor A, cyclin-dependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4), INK4, INK4a, MLMP16INK4, MTS-1, MTS1P14, Multiple tumor suppressor 1, p14, p14ARF, P16, p16-INK4, p16INK4a, p16-INK4a, P19, P19ARF, TP16

Entrez Gene IDs

1029 (Human)

Gene Symbol

CDKN2A

UniProt

Q8N726 (Human)

Additional p14ARF/CDKN2A Products

Product Documents for p14ARF/CDKN2A Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

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Product Specific Notices for p14ARF/CDKN2A Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for p14ARF/CDKN2A Antibody - BSA Free

Customer Reviews for p14ARF/CDKN2A Antibody - BSA Free (2)

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2 Customer Ratings
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  • p14ARF/CDKN2A Antibody
    Name: Ming Hua
    Application: Western Blot
    Sample Tested: OCI/AML3 cells and human AML NPM cytoplasmic mutant cell line
    Species: Human
    Verified Customer | Posted 09/01/2017
    ARF expression levels in OCI/AML3 cells after proteasome treatment
    p14ARF/CDKN2A Antibody - BSA Free NB200-111
  • Name: Ronald Miller
    Application: Western Blot
    Sample Tested: Young and senescent cells
    Species: Human
    Verified Customer | Posted 05/29/2014
    p14/ARF levels in yourng and senescent cells
    p14ARF/CDKN2A Antibody - BSA Free NB200-111

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Protocols

View specific protocols for p14ARF/CDKN2A Antibody - BSA Free (NB200-111):


Western Blot Procedure

1) Lyse HeLa or BT549 cells in Laemli buffer (2% SDS, 62.5 mM Tris pH 6.8, 10% glycerol, 5% 2-mercaptoethanol).
2) Boil the lysate for 5 minutes.
3) Load 25 ug of total cell extract, per lane, in a 15% SDS-PAGE minigel.
4) Transfer protein to PVDF membrane in 40 mM Tris base, 20 mM NaAcetate, 2 mM EDTA pH 7.4, 0.05% SDS, 20% methanol for 1 hour at 4 degrees C.
5) Rinse membrane in PBS-T and then block membrane in 5% nonfat dry milk/PBS-T (0.01M phosphate, 0.0027M KCl, 0.137M NaCl pH 7.4, 0.1% Tween-20) for 1 hour at room temperature or overnight at 4 degrees C.
6) Incubate membrane overnight at 4 degrees C with properly diluted NB200-111 (see data sheet) in PBS, 0.2% Tween-20, 5% nonfat dry milk.
7) Rinse the membrane 2X with 40 ml PBS-T. Wash membrane at room temperature, with 40 ml of PBS-T, 1X for 15 minutes and 2X for 5 minutes, each.
8) Incubate membrane with HRP conjugated anti-rabbit, diluted in PBS-T with 5% nonfat dry milk, for 1 hour at room temperature.
9) Wash membrane at room temperature, with 40 ml of PBS-T, 1X for 15 minutes and 4X for 5 minutes, each.
10) Develop with ECL reagents (Amersham) and autoradiography. Expose for 1+ minutes. For BT549 cells, 1 minute exposure is needed. For HeLa cells, additional exposure time may be required. In addition, a stronger ECL (Pierce) may be necessary.

NOTE: HeLa whole cell extracts (NB800-PC1) were used as a positive control for this antibody.

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for p14ARF/CDKN2A Antibody - BSA Free

Showing  1 - 2 的 2 FAQs Showing All

    • Q: Do you measure the isotype purity of NB200-111? I realize this is antigen affinity purified, and will be almost completely IgG, but do you measure for IgA/M amount by chance?
      A: We unfortunately do not measure isotype purity for our products.

    • Q: While looking for antibodies targeting human p14ARF, I found a question: Based on the information on UniProt, human p14ARF has the length of only 132 a.a. However, the immunogen of NB200-159 is described as... between residue 125 and the C-terminus (residue 173)...,...between residues 100-173... for NB110-59085, and...between residues 50-150... for NB200-111.
      A: Since these antibodies were produced, UniProt has since truncated the protein sequence. These antibodies are still directed towards p14ARF and the immunogens will be updated on our website to the updated amino acid ranges. Since you won't be able to see this until this weekend or early next week, the immunogen for NB200-111 falls between amino acids 50 and 132. The immunogen for NB200-159 falls between amino acids 90 and 132 and the immunogen for NB110-59085 falls between amino acids 50 and 132.
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