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.
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 HCT 116 cell lysate
Lane 2 : CDKN1A knockout HCT 116 cell lysate
Lane 3 : Wild-type MCF7 cell lysate
Lane 4 : CDKN1A knockout MCF7 cell lysate
Lane 5 : Wild-type A549 cell lysate
Lane 6 : CDKN1A knockout A549 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 21 kDaWestern blot: Anti-CDKN1A antibody [EPR362] (ab109520) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to CDKN1A. A band was observed at 21 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CDKN1A knockout cell line. To generate this image, wild-type and CDKN1A knockout HCT 116 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution
Lane 1 : wild-type HeLa Vehicle Control Fluvastatin (20 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 (20 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) 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) 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) 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 (331)
ab109520 has been referenced in 331 publications.
- Tryfonos A et al. Association between atherogenic risk-modulating proteins and endothelium-dependent flow-mediated dilation in coronary artery disease patients. Eur J Appl Physiol 123:367-380 (2023). PubMed: 36305972
- Ni T et al. PTBP1 drives c-Myc-dependent gastric cancer progression and stemness. Br J Cancer 128:1005-1018 (2023). PubMed: 36635500
- Shen E et al. DEP domain containing 1B (DEPDC1B) exerts the tumor promoter in hepatocellular carcinoma through activating p53 signaling pathway via kinesin family member 23 (KIF23). Bioengineered 13:1103-1114 (2022). PubMed: 34983303
- Li R et al. IMP4 Silencing Inhibits the Malignancy of Lung Adenocarcinoma via ERK Pathway. J Oncol 2022:8545441 (2022). PubMed: 36317123
- Tian C et al. Blockade of FGF2/FGFR2 partially overcomes bone marrow mesenchymal stromal cells mediated progression of T-cell acute lymphoblastic leukaemia. Cell Death Dis 13:922 (2022). PubMed: 36333298