Overview

  • Product name
    Human p21 ELISA Kit
    See all p21 kits
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    MCF7 lysates 5 4.2%
    Inter-assay
    Sample n Mean SD CV%
    MCF7 lysates 3 7.3%
  • Sample type
    Cell culture extracts, Tissue Lysate
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    11 pg/ml
  • Range
    62.5 pg/ml - 4000 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 120 115% - 129%

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    Human p21 beta SimpleStep ELISA® kit (ab171579) has been re-developed with new capture and detector antibodies. This new kit has the same name but a different product number (ab214568). We have identified new recombinant monoclonal antibodies to use in the SimpleStep ELISA platform that provide higher sensitivity when quantification of p21 in human cell culture extracts and tissue lysates.


    p21 in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of p21 protein in human cell and tissue extracts.


    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    p21 plays a critical role in the cellular response to DNA damage, and its overexpression results in cell cycle arrest. Upregulation of p21 mRNA and protein following ionizing radiation is dependent on p53 (TP53), and p21 mediates cell cycle arrest in response to the p53 checkpoint pathway. p21 binds to and inhibits cyclin-dependent kinase activity, preventing phosphorylation of critical cyclin-dependent kinase substrates and blocking cell cycle progression. p21 functions in the nuclear localization and assembly of cyclin D-CDK4 complex and promotes its kinase activity towards RB1. At higher stoichiometric ratios, p21 inhibits the kinase activity of the cyclin D-CDK4 complex.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X p21 Capture Antibody 1 x 600µl
    10X p21 Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml
    Antibody Diluent 5BI 1 x 6ml
    p21 Human Lyophilized Recombinant Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS (ab193972) 1 x 12ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 x 96 tests
    Stop Solution 1 x 12ml
    TMB Substrate 1 x 12ml
  • Research areas
  • 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
  • Alternative names
    • CAP20
    • CDK-interacting protein 1
    • CDKI
    • CDKN1
    • Cdkn1a
    • CDN1A_HUMAN
    • CIP1
    • Cyclin Dependent Kinase Inhibitor 1A
    • Cyclin-dependent kinase inhibitor 1
    • Cyclin-dependent kinase inhibitor 1A (P21)
    • Cyclin-dependent kinase inhibitor 1A (p21, Cip1)
    • DNA Synthesis Inhibitor
    • MDA-6
    • MDA6
    • Melanoma differentiation-associated protein
    • Melanoma differentiation-associated protein 6
    • p21
    • P21 protein
    • p21CIP1
    • p21Cip1/Waf1
    • p21WAF
    • PIC1
    • SDI1
    • SLC12A9
    • WAF1
    • Wild type p53 activated fragment 1
    • Wild type p53 activated fragment 1 (WAF1)
    • Wildtype p53-activated fragment 1
    see all
  • Database links

Associated products

Applications

Our Abpromise guarantee covers the use of ab214658 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
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of p21 were measured in duplicates in a two-fold dilution series and interpolated from the p21 standard curve and corrected for sample dilution.  The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).  The mean p21 concentration was determined to be 1,811 pg/mL in extract sample of MCF-7 cells treated for 18 hours with 1 mM camptothecin.

  • Interpolated concentrations of native p21 in human extract samples of HeLa cells, vehicle (DMSO)-treated MCF-7 cells and 18 hours 1 µM camptothecin-treated MCF-7 cells samples.  The concentrations of p21 were measured in duplicates at 3 different dilutions, interpolated from the p21 standard curve. Note that Undiluted HeLa cell extract and Undiluted vehicle (DMSO)-treated MCF-7cell extract samples were at 32 g/mL. Note that Undiluted 18 hours 1 M camptothecin-treated MCF-7 cells samples were at 8 µg/mL. The interpolated, dilution factor-corrected values are graphed in pg of p21 per µg of total protein (mean +/- SD, n=2).

Protocols

References

ab214658 has not yet been referenced specifically in any publications.

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