Knockout Tested Rabbit Recombinant Monoclonal p21 antibody. Suitable for IHC-P, IP, WB, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 390 publications.
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
IHC-P | IP | WB | ICC/IF | Flow Cyt (Intra) | |
---|---|---|---|---|---|
Human | Tested | Tested | Tested | Tested | Tested |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/100 - 1/250 | Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/10 - 1/100 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 - 1/10000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 | Notes For unpurified use at 1/50 - 1/100. |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/100 | Notes - |
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May be involved in p53/TP53 mediated inhibition 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. Inhibits DNA synthesis by DNA polymerase delta by competing with POLD3 for PCNA binding (PubMed:11595739). Plays an important role in controlling cell cycle progression and DNA damage-induced G2 arrest (PubMed:9106657).
Cyclin-dependent kinase inhibitor 1, CDK-interacting protein 1, Melanoma differentiation-associated protein 6, p21, MDA-6, CDKN1A, MDA6, CAP20, CDKN1, CIP1, PIC1, SDI1, WAF1
Knockout Tested Rabbit Recombinant Monoclonal p21 antibody. Suitable for IHC-P, IP, WB, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 390 publications.
Cyclin-dependent kinase inhibitor 1, CDK-interacting protein 1, Melanoma differentiation-associated protein 6, p21, MDA-6, CDKN1A, MDA6, CAP20, CDKN1, CIP1, PIC1, SDI1, WAF1
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EPR362
Affinity purification Protein A
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.
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Stable for 12 months at -20°C
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
The p21 protein also known as CDKN1A is an important regulator of cell cycle progression. It acts as a cyclin-dependent kinase inhibitor where it binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes. The molecular weight of p21 is approximately 21 kDa. Cellular expression of p21 is diverse; it occurs in various tissues reflecting its broad role in maintaining cell cycle control. This protein's expression level is often assessed using techniques such as p21 western blotting p21 ELISA and p21 IHC staining.
P21 plays a critical role in cell cycle regulation and DNA damage response. It associates with the p53 tumor suppressor protein forming an essential part of the p53 signaling pathway under stress conditions. p21 arrests cells in G1 phase allowing DNA repair or leading to cellular senescence. Through these mechanisms p21 integrates signals from stress and damage emphasizing its importance as a mediator in cellular checkpoint control.
P21 is closely involved in the p53 and the Transforming Growth Factor-beta (TGF-β) signaling pathways. This integration helps in transmitting growth-inhibitory signals. p21 interacts with proteins such as p53 and cyclins ensuring precise regulation of the cell cycle and growth arrest when necessary. Triggered by p53 activation p21 contributes to preventing undisturbed cell proliferation acting as a safeguard against tumor formation.
P21's deregulation associates with cancer and age-related diseases. Its impairment is a common feature in many cancers often linked with defects in the p53 pathway. Reduced p21 expression can contribute to unchecked cell division and tumor progression. Moreover in age-related diseases p21's role in promoting cellular senescence connects it with degenerative conditions. The intricate relationship between p21 and proteins such as p53 and cyclins highlights its potential as a therapeutic target for these disorders.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
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Terms & Conditions.
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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab109520 in HEK293 whole cell lysate.
For western blotting, VeriBlot for IP Detection Reagent (HRP) (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.
All lanes: Immunoprecipitation - Anti-p21 antibody [EPR362] (ab109520)
Predicted band size: 18 kDa
Observed band size: 21 kDa
False colour image of Western blot: Anti-p21 antibody [EPR362] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 Human CDKN1A knockout HeLa 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution
Lane 1: wild-type HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg
Lane 2: wild-type HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg
Lane 3: CDKN1A knockout HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg
Lane 4: CDKN1A knockout HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg
Lane 5: MCF7 cell lysate at 20 µg
Lane 6: SH-SY5Y cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 18 kDa
Observed 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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520)
Predicted band size: 18 kDa
Observed band size: 20 kDa
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. Goat Anti-Rabbit IgG H&L (HRP) 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.
Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) labeling p21 with ab109520 at 1/500 (2 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, Alexa Fluor®488 Goat anti-Rabbit at 1/1000 (2 μg/ml) was used as secondary antibody. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.
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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.
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.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520)
Predicted band size: 18 kDa
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. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.
Control 1: primary antibody (1/1000) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).
Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).
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, Anti-GAPDH antibody [6C5] - 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: Western blot - Anti-p21 antibody [EPR362] (ab109520)
Predicted band size: 18 kDa
Blocking and dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/2000 dilution
Lane 1: MCF7 cell lysate at 20 µg
Lane 2: HEK293 cell lysate at 20 µg
Lane 3: U87-MG cell lysate at 20 µg
All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution
Predicted band size: 18 kDa, 24 kDa, 57 kDa
Observed band size: 21 kDa
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.
Blocking and dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/10000 dilution
All lanes: LnCaP cell lysate at 20 µg
All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution
Predicted band size: 18 kDa
Observed band size: 21 kDa
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.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution
Lane 1: MCF7 cell lysate at 10 µg
Lane 2: HeLa cell lysate at 10 µg
Lane 3: HUVEC cell lysate at 10 µg
Lane 4: LnCap cell lysate at 10 µg
Lane 5: U87 MG cell lysate at 10 µg
Lane 6: 293T cell lsyate at 10 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab6721)
Predicted band size: 18 kDa
Western blot: Anti-CDKN1A antibody [EPR362] (ab109520) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control 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.
All lanes: Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: CDKN1A knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 cell lysate at 20 µg
Lane 4: CDKN1A knockout MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 cell lysate at 20 µg
Lane 6: CDKN1A knockout A549 cell lysate at 20 µg
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 21 kDa
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