Overview

  • Product name
    Anti-p21 antibody [EPR362]
    See all p21 primary antibodies
  • Description
    Rabbit monoclonal [EPR362] to p21
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, WB, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human p21 aa 1-100. The exact sequence is proprietary.

  • Positive control
    • WB: MCF7, HeLa, HEK293, HUVEC, LnCaP, U87 MG or 293T cell lysates. IHC-P: Human cervical carcinoma or papillary carcinoma of thyroid gland tissues. ICC/IF: MCF-7 cells. Flow Cyt: HeLa cells. IP: HEK293 cell lysate.
  • General notes

    A trial size is available to purchase for this antibody.

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab109520 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
ICC/IF 1/1000.

For unpurified use at 1/50 - 1/100.

Flow Cyt 1/100.
WB 1/1000 - 1/10000. Predicted molecular weight: 21 kDa.
IP 1/10 - 1/100.
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

Target

  • Function
    May be the important intermediate by which p53/TP53 mediates its role as an inhibitor of cellular proliferation in response to DNA damage. Binds to and inhibits cyclin-dependent kinase activity, preventing phosphorylation of critical cyclin-dependent kinase substrates and blocking cell cycle progression. Functions in the nuclear localization and assembly of cyclin D-CDK4 complex and promotes its kinase activity towards RB1. At higher stoichiometric ratios, inhibits the kinase activity of the cyclin D-CDK4 complex.
  • Tissue specificity
    Expressed in all adult human tissues, with 5-fold lower levels observed in the brain.
  • Sequence similarities
    Belongs to the CDI family.
  • Domain
    The PIP-box K+4 motif mediates both the interaction with PCNA and the recuitment of the DCX(DTL) complex: while the PIP-box interacts with PCNA, the presence of the K+4 submotif, recruits the DCX(DTL) complex, leading to its ubiquitination.
    The C-terminal is required for nuclear localization of the cyclin D-CDK4 complex.
  • Post-translational
    modifications
    Phosphorylation of Thr-145 by Akt or of Ser-146 by PKC impairs binding to PCNA. Phosphorylation at Ser-114 by GSK3-beta enhances ubiquitination by the DCX(DTL) complex.
    Ubiquitinated by MKRN1; leading to polyubiquitination and 26S proteasome-dependent degradation. Ubiquitinated by the DCX(DTL) complex, also named CRL4(CDT2) complex, leading to its degradation during S phase or following UV irradiation. Ubiquitination by the DCX(DTL) complex is essential to control replication licensing and is PCNA-dependent: interacts with PCNA via its PIP-box, while the presence of the containing the 'K+4' motif in the PIP box, recruit the DCX(DTL) complex, leading to its degradation.
  • Cellular localization
    Cytoplasm. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • CAP20 antibody
    • CDK-interacting protein 1 antibody
    • CDKI antibody
    • CDKN1 antibody
    • Cdkn1a antibody
    • CDN1A_HUMAN antibody
    • CIP1 antibody
    • Cyclin Dependent Kinase Inhibitor 1A antibody
    • Cyclin-dependent kinase inhibitor 1 antibody
    • Cyclin-dependent kinase inhibitor 1A (P21) antibody
    • Cyclin-dependent kinase inhibitor 1A (p21, Cip1) antibody
    • DNA Synthesis Inhibitor antibody
    • MDA-6 antibody
    • MDA6 antibody
    • Melanoma differentiation-associated protein 6 antibody
    • Melanoma differentiation-associated protein antibody
    • p21 antibody
    • P21 protein antibody
    • p21CIP1 antibody
    • p21Cip1/Waf1 antibody
    • p21WAF antibody
    • PIC1 antibody
    • SDI1 antibody
    • SLC12A9 antibody
    • WAF1 antibody
    • Wild type p53 activated fragment 1 (WAF1) antibody
    • Wild type p53 activated fragment 1 antibody
    • Wildtype p53-activated fragment 1 antibody
    see all

Anti-p21 antibody [EPR362] images

  • Cell line MCF7 (Human breast adenocarcinoma cell line), Target AbID ab109520  anti-p21 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary. Counterstain AbID ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Fixative 4% Paraformaldehyde, Permeabilisation 0.1% tritonX-100, Nuclear counter stain DAPI. Comments Confocal image showing nuclear staining on MCF7 cell line. Target 1oAb dilution 1:500 2 μg/ml, Target 2ndry Ab dilution 1:1000 2 μg/ml, Counterstain Ab dilution 1:200 2.5 μg/ml.

     



  • Predicted band size : 21 kDa
    Observed band size : 20 kDa (why is the actual band size different from the predicted?)

    Lane 1: Wild-type DLD-1 cell lysate (20 µg)

    Lane 2: Wild-type DLD-1 20 μM 2,3-DCPE for 16hrs treated cell lysate (20 µg)

    Lane 3: p21 knockout DLD-1 cell lysate (20 µg)

    Lane 4: p21 knockout 20 μM 2,3-DCPE for 16hrs DLD-1 cell lysate (20 µg)

    Lane 5: HT1080 cell lysate (20 μg)


    Lanes 1 - 5: Merged signal (red and green). Green - ab109520 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109520 was shown to specifically recognize p21 when p21 knockout samples were used. Wild-type and p21 knockout samples were subjected to SDS-PAGE. ab109520 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  • Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling p21 with purified ab109520 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.

    Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling p21 with purified ab109520 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Overlay histogram showing HeLa cells stained with unpurified ab109520 (red line). The cells were fixed with 80% methanol (5 min) then permeabilized with 0.1% PBS-Tween 20 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 (unpurified ab109520, 1/100) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

    Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.



  • Predicted band size : 21 kDa

    Lane 1: Wild-type DLD-1 cell lysate (20 µg)
    Lane 2: Wild-type DLD-1 20 μM 2,3-DCPE for 16hrs treated cell lysate (20 µg)
    Lane 3: p21 knockout DLD-1 cell lysate (20 µg)
    Lane 4: p21 knockout 20 μM 2,3-DCPE for 16hrs DLD-1 cell lysate (20 µg)
    Lane 5: HT1080 cell lysate (20 μg)

    Lanes 1 - 5: Merged signal (red and green). Green - ab109520 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab109520 and a competitor's top cited rabbit polyclonal antibody.

  • All lanes : Anti-p21 antibody [EPR362] (ab109520) at 1/2000 dilution (purified)

    Lane 1 : MCF7 cell lysate
    Lane 2 : HEK293 cell lysate
    Lane 3 : U87-MG cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

    Predicted band size : 21 kDa
    Observed band size : 21 kDa

    Blocking and dilution buffer: 5% NFDM/TBST.

  • ab109520 (purified) at 1/50 immunoprecipitating p21 in HEK293 whole cell lysate.

    Lane 1 (input): HEK293 whole cell lysate (10µg)

    Lane 2 (+): ab109520 + HEK293 whole cell lysate (10µg).

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109520 in HEK293 whole cell lysate.

    For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1500).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human papillary carcinoma of the thyroid gland tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

  • Anti-p21 antibody [EPR362] (ab109520) at 1/10000 dilution (purified) + LnCaP cell lysate at 20 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

    Predicted band size : 21 kDa
    Observed band size : 21 kDa

    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

  • All lanes : Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution (unpurified)

    Lane 1 : MCF7 cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HUVEC cell lysate
    Lane 4 : LnCap cell lysate
    Lane 5 : U87 MG cell lysate
    Lane 6 : 293T cell lsyate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721)

    Predicted band size : 21 kDa

References for Anti-p21 antibody [EPR362] (ab109520)

This product has been referenced in:
  • Zhang Z  et al. Sepia ink oligopeptide induces apoptosis and growth inhibition in human lung cancer cells. Oncotarget 8:23202-23212 (2017). WB ; Human . Read more (PubMed: 28423568) »
  • Lin X  et al. Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells. Oncol Lett 13:1695-1701 (2017). WB ; Human . Read more (PubMed: 28454311) »

See all 17 Publications for this product

Product Wall

Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Specification
MEF
Blocking step
ODYSSEY BLOCKING BUFFER as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C
Username

Muhammed Zoabi

Verified customer

Submitted Apr 27 2017

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Sample
Human Cell (MCF-7)
Specification
MCF-7
Permeabilization
Yes - Triton X-100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 04 2014

Application
Western blot
Loading amount
50000 cells
Gel Running Conditions
Reduced Denaturing (10% gel)
Sample
Human Cell lysate - whole cell (MCF-7)
Specification
MCF-7
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Aug 04 2014

Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Reduced Denaturing (13%)
Sample
Human Cell lysate - whole cell (HeLa, HCT116 and MEF cell lysates)
Specification
HeLa, HCT116 and MEF cell lysates
Treatment
DMSO control (Ctl) and 7,5 ´M CPT-11 (CPT) for 24 hrs
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Christian Marx

Verified customer

Submitted Jan 15 2014

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