Overview

  • Product nameAnti-p21 antibodySee all p21 primary antibodies ...
  • Description
    Rabbit polyclonal to p21
  • SpecificityDetects a clean band at 21kDa corresponding to p21, as well as a cross-reacting band at 26kDa.
  • Tested applicationsICC/IF, Sandwich ELISA, WB, IHC-P more details
  • Species reactivity
    Reacts with: Rat, Cow, Human
    Predicted to work with: Cat, Dog
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human p21.

    (Peptide available as ab18623.)

  • Positive control
    • This antibody gave a positive signal in the following whole cell lysates: HeLa; Hela - Bleomycin Treated (40U/ml); HeLa Whole - Irradiated (5Gy); Hela - Hydroxyurea Treated (48hr, 1uM); PC12.

Properties

Applications

Our Abpromise guarantee covers the use of ab18209 in the following tested applications.

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

Application Notes
ICC/IF
Sandwich ELISA
WB
IHC-P
  • Application notesIHC-P: Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
    ICC/IF: Use at a concentration of 1 µg/ml.
    WB: Use at a concentration of 1 µg/ml. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa).


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • FunctionMay 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 specificityExpressed in all adult human tissues, with 5-fold lower levels observed in the brain.
    • Sequence similaritiesBelongs to the CDI family.
    • DomainThe 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 localizationCytoplasm. Nucleus.
    • Target information above from: UniProt accession P38936 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links
    • Alternative names
      • Activating Fragment 1 antibody
      • Activating Fragment 1 antibody
      • CAP20 antibody
      • CAP20 antibody
      • Cation chloride cotransporter-interacting protein 1 antibody
      • CDK Interacting Protein 1 antibody
      • CDK Interacting Protein 1 antibody
      • CDK-interacting protein 1 antibody
      • CDKI antibody
      • CDKN 1 antibody
      • CDKN1 antibody
      • CDKN1 antibody
      • CDKN1A antibody
      • CDKN1A antibody
      • CDN1A_HUMAN antibody
      • CIP1 antibody
      • CIP1 antibody
      • Cyclin Dependent Kinase Inhibitor 1A 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
      • MDA-6 antibody
      • MDA6 antibody
      • MDA6 antibody
      • Melanoma Differentiation Associated Protein 6 antibody
      • Melanoma differentiation-associated protein 6 antibody
      • Melanoma differentiation-associated protein antibody
      • p21 antibody
      • p21 antibody
      • P21 protein antibody
      • p21CIP1 antibody
      • p21Cip1/Waf1 antibody
      • p21WAF antibody
      • PIC1 antibody
      • PIC1 antibody
      • SDI1 antibody
      • SDI1 antibody
      • SLC12A9 antibody
      • WAF1 antibody
      • WAF1 antibody
      • Wild type p53 activated fragment 1 antibody
      • Wildtype p53 Activating Fragment 1 antibody
      • Wildtype p53 Activating Fragment 1 antibody
      • Wildtype p53-activated fragment 1 antibody
      see all

    Anti-p21 antibody images

    • IHC image of p21 staining in human lung FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18209, 5ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    • ab18209 staining p21 in serum starved PC-3 cells treated with β-lapachone (ab141097), by ICC/IF. Increase of p21 nuclear expression correlates with increased concentration of β-lapachone, as described in literature.
      The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141097 (β-lapachone) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab18209 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
    • ICC/IF image of ab18209 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18209, 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).

    • The image shows staining of human embryonic stem cells with ab18209. Staining appears to be nuclear.
    • All lanes : Anti-p21 antibody (ab18209) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : HeLa Whole Cell Lysate - Irradiated (5Gy)
      Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
      developed using the ECL technique

      Performed under reducing conditions.

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


      Exposure time : 30 seconds

      This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab18209 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

    • Anti-p21 antibody (ab18209) at 1 µg/ml + HeLa whole cell lysate at 20 µg/ml

      Secondary
      Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/10000 dilution

      Predicted band size : 21 kDa
      Observed band size : 21 kDa
      Additional bands at : 26 kDa (possible cross reactivity).
    • All lanes : Anti-p21 antibody (ab18209) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : Hela Whole Cell Lysate - Bleomycin Treated (40U/ml)
      Lane 3 : HeLa Whole Cell Lysate - Irradiated (5Gy)
      Lane 4 : Hela Whole Cell Lysate - Hydroxyurea Treated (48hr, 1uM)

      Lysates/proteins at 20 µg per lane.

      Secondary
      Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
      developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 21 kDa
      Observed band size : 21 kDa
      Additional bands at : 36 kDa,56 kDa. We are unsure as to the identity of these extra bands.

      Exposure time : 20 minutes
    • Standard curve for p21 (Analyte: ab56278); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [AC8.3] to p21 (ab118) at 5µg/ml and Detector Antibody Rabbit polyclonal to p21 (ab18209) at 0.5µg/ml.
    • Anti-p21 antibody (ab18209) at 1 µg/ml + Human p21 full length protein (ab56278) at 0.01 µg

      Secondary
      Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/5000 dilution
      developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 21 kDa


      Exposure time : 150 seconds
      ab56278 is a tagged recombinant protein, with a predicted molecular weight of 46kDa.

    Protocols

    References for Anti-p21 antibody (ab18209)

    This product has been referenced in:
    • Sousa MM  et al. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells. PLoS One 8:e55493 (2013). WB ; Human . Read more (PubMed: 23405159) »
    • Symanowski J  et al. A histone deacetylase inhibitor LBH589 downregulates XIAP in mesothelioma cell lines which is likely responsible for increased apoptosis with TRAIL. J Thorac Oncol 4:149-60 (2009). Read more (PubMed: 19179889) »

    See all 2 Publications for this product

    Product Wall

    Thank you for your reply.

    While I did not receive the image which you have referenced I would like to offer so help regarding your questions. Generally specific details regarding secondary antibody incubation and concentration as will need...

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    Thank you for helping my confusion about which image we were discussing. This information does help to understand the results you are getting with this product. These non specific bands have not been identified ho...

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    I will forward your questions to the lab. However I can answer a few. We used 20ug/well of protein. Our standard protocol is to run transfers run wet or semi-dry for 40minutes to 1 hour. Generally we use chemil...

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    I am very sorry to hear that you have been experiencing difficulties when using this product with the recommended HeLa whole cell lysate positive control. We have achieved best results when using this antibody...

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    Application Western blot
    Sample Cow Cell lysate - whole cell (aortic endothelial cells)
    Loading amount 20 µg
    Specification aortic endothelial cells
    Treatment 10uM 4HNE for 5 days
    Gel Running Conditions Reduced Denaturing (15% gel)
    Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Username

    Abcam user community

    Verified customer

    Submitted Feb 03 2011

    Application Immunocytochemistry/ Immunofluorescence
    Sample Human Cell (Fibroblasts)
    Specification Fibroblasts
    Fixative Paraformaldehyde
    Permeabilization Yes - 0.5% Triton-X
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 21°C
    Username

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    Verified customer

    Submitted May 25 2010

    Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample Human Tissue sections (Skin, verruca vulgaris (wart))
    Specification Skin, verruca vulgaris (wart)
    Fixative Formaldehyde
    Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer pH 6.0
    Permeabilization Yes - TBST
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
    Username

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    Verified customer

    Submitted Feb 16 2010

    Thank you for contacting us for technical support, I'm sorry to hear you are having problems with ab18209 and am worried the high band you are seeing is the non specific band we see at 29kda (please see image). I cannot offer you a free sample of th...

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