Recombinant Anti-p21 antibody [EPR362] (ab109520)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR362] to p21
- Suitable for: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-p21 antibody [EPR362]
See all p21 primary antibodies -
Description
Rabbit monoclonal [EPR362] to p21 -
Host species
Rabbit -
Specificity
Expression levels of the target protein vary between different tissue/cell lines and in some cases induction may be required before a signal is observed. -
Tested applications
Suitable for: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-Pmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: MCF7, HeLa, HEK293, HUVEC, LnCaP, U87 MG or HEK-293T cell lysates. IHC-P: Human cervical carcinoma or papillary carcinoma of thyroid gland tissues. ICC/IF: MCF-7 cells. Flow Cyt (intra): HeLa cells. IP: HEK-293 cell lysate.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol, 59% PBS, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR362 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
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 |
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Flow Cyt (Intra) |
1/100.
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ICC/IF | (3) |
1/1000.
For unpurified use at 1/50 - 1/100. |
WB | (5) |
1/1000 - 1/10000. Predicted molecular weight: 21 kDa.
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IP |
1/10 - 1/100.
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IHC-P | (3) |
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Notes |
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Flow Cyt (Intra)
1/100. |
ICC/IF
1/1000. For unpurified use at 1/50 - 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. |
Target
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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
modificationsPhosphorylation 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
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Database links
- Entrez Gene: 1026 Human
- Omim: 116899 Human
- SwissProt: P38936 Human
- Unigene: 370771 Human
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Alternative names
- CAP20 antibody
- CDK-interacting protein 1 antibody
- CDKI antibody
see all
Images
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All lanes : Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution
Lane 1 : wild-type HeLa Vehicle Control Fluvastatin (0 uM, 24 h) cell lysate
Lane 2 : wild-type HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate
Lane 3 : CDKN1A knockout HeLa Vehicle Control Fluvastatin (0 uM, 24 h) cell lysate
Lane 4 : CDKN1A knockout HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate
Lane 5 : MCF7 cell lysate
Lane 6 : SH-SY5Y cell lysate
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 21 kDaFalse colour image of Western blot: Anti-p21 antibody [EPR362] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to p21. A band was observed at 21 kDa in wild-type HeLa cell lysates with no signal observed at this size in CDKN1A knockout cell line ab255349 (knockout cell lysate ab263812). To generate this image, wild-type and CDKN1A knockout cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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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 in wild-type DLD-1 cells treated with 20 μM 2,3-DCPE. No band was observed when p21 knockout samples +/- 2,3-DCPE treatment 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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] (ab109520)
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.
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Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) labeling p21 with ab109520 at 1/500 (2 μg/ml). ab150077, Alexa Fluor®488 Goat anti-Rabbit at 1/1000 (2 μg/ml) was used as secondary antibody. ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 (2.5 μg/ml) was used as counterstain AbID. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Nuclei were counter stained blue with DAPI.
Confocal image showing nuclear staining on MCF7 cell line.
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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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
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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 22°C. The secondary antibody used was Alexa Fluorr® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. 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.
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Different batches of ab109520 were tested on MCF7 (Human breast adenocarcinoma epithelial cell) lysate at 0.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 21 kDa.
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Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labeling 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).
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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.
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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
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDaBlocking and dilution buffer: 5% NFDM/TBST.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] (ab109520)
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.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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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 kDaBlocking and dilution buffer: 5% NFDM/TBST.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] (ab109520)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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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
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721)
Predicted band size: 21 kDa
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (257)
ab109520 has been referenced in 257 publications.
- Zhang Y et al. Long Non-coding RNA CASC15 Promotes Intrahepatic Cholangiocarcinoma Possibly through Inducing PRDX2/PI3K/AKT Axis. Cancer Res Treat 53:184-198 (2021). PubMed: 33017884
- Niu L et al. Sericin inhibits MDA-MB-468 cell proliferation via the PI3K/Akt pathway in triple-negative breast cancer. Mol Med Rep 23:N/A (2021). PubMed: 33313947
- Yang Y & Min Z Effect of long non-coding RNA AK021443 on promoting hepatic fibrosis in vitro. Mol Med Rep 23:N/A (2021). PubMed: 33495828
- Ji H et al. SP1 induced long non-coding RNA AGAP2-AS1 promotes cholangiocarcinoma proliferation via silencing of CDKN1A. Mol Med 27:10 (2021). PubMed: 33522895
- Wang H et al. Knockdown of Dual Oxidase 1 (DUOX1) Promotes Wound Healing by Regulating Reactive Oxygen Species (ROS) by Activation of Nuclear Kactor kappa B (NF-?B) Signaling. Med Sci Monit 27:e926492 (2021). PubMed: 33563887