Anti-p21 antibody [EPR362] - Carrier free (ab218311)

Anti-p21 [EPR362] antibody (ab218311) is a rabbit monoclonal antibody that is provided in a PBS only formulation free from BSA and azide. Formulated to be conjugation-ready. It is used to detect p21 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IP, IHC-P, ICC/IF. Suitable for Human samples.

- Specificity confirmed with p21 knockout cell line validation

-BSA,?sodium azide, and glycerol-free?for consistent conjugation with fluorochromes, enzymes and more

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Images

Immunoprecipitation - Anti-p21 antibody [EPR362] - BSA and Azide free (AB218311), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IHC-P	IP	WB	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Associated Products

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Target data

See full target information CDKN1A

Function

Plays an important role in controlling cell cycle progression and DNA damage-induced G2 arrest (PubMed:9106657). Involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Also involved in p53-independent DNA damage-induced G2 arrest mediated by CREB3L1 in astrocytes and osteoblasts (By similarity). 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). Negatively regulates the CDK4- and CDK6-driven phosphorylation of RB1 in keratinocytes, thereby resulting in the release of E2F1 and subsequent transcription of E2F1-driven G1/S phase promoting genes (By similarity).

Alternative names

CAP20, CDKN1, CIP1, MDA6, PIC1, SDI1, WAF1, CDKN1A, Cyclin-dependent kinase inhibitor 1, CDK-interacting protein 1, Melanoma differentiation-associated protein 6, p21, MDA-6

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Carrier free: Yes
Clone number: EPR362
Purification technique: Affinity purification Protein A
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.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: +4°C
Storage information: Do Not Freeze

Notes

ab218311 is the carrier-free version of Anti-p21 antibody [EPR362] ab109520.

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
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 batch production

For more information, read more on recombinant antibodies.

Conjugation ready
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

Compatibility
This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

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.

Biological function summary

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.

Pathways

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.

Associated diseases and disorders

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.

Product promise

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10 product images

Immunoprecipitation - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Anti-p21 antibody [EPR362] ab109520 (purified) at 1/50 immunoprecipitating p21 in HEK293 whole cell lysate.
Lane 1 (input): HEK293 whole cell lysate (10μg)
Lane 2 (+): Anti-p21 antibody [EPR362] ab109520 + HEK293 whole cell lysate (10μg).
Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-p21 antibody [EPR362] ab109520 in HEK293 whole cell lysate.
For western blotting, VeriBlot for IP Detection Reagent (HRP) ab131366 VeriBlot for IP (HRP) was used for detection at 1/1500 dilution.
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
All lanes: Immunoprecipitation - Anti-p21 antibody [EPR362] (Anti-p21 antibody [EPR362] ab109520)
Predicted band size: 18 kDa
Observed band size: 21 kDa
Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
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, Anti-p21 antibody [EPR362] 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 y 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
All lanes: Western blot - Anti-p21 antibody [EPR362] (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
Lanes 3 - 4: Western blot - Human CDKN1A knockout HeLa cell line (Human CDKN1A knockout HeLa cell line ab255349)
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
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling p21 with purified Anti-p21 antibody [EPR362] 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Cell line MCF7 (Human breast adenocarcinoma cell line), Target AbID Anti-p21 antibody [EPR362] ab109520 anti-p21 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor®488 Goat anti-Rabbit secondary. Counterstain AbID Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Fixative 4% Paraformaldehyde, Permeabilisation 0.1% tritonX-100, Nuclear counter stain DAPI. Comments Confocal image showing nuclear staining on MCF7 cell line. Target 1oAb dilution 1:500 2 μg/ml, Target 2ndry Ab dilution 1:1000 2 μg/ml, Counterstain Ab dilution 1:200 2.5 μg/ml.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Flow Cytometry (Intracellular) - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Overlay histogram showing HeLa cells stained with unpurified Anti-p21 antibody [EPR362] 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 Anti-p21 antibody [EPR362] ab109520, 1/100) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
This data was developed using Anti-p21 antibody [EPR362] ab109520, the same antibody clone in a different buffer formulation. Different batches of Anti-p21 antibody [EPR362] 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] (Anti-p21 antibody [EPR362] ab109520)
Predicted band size: 18 kDa
Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling p21 with purified Anti-p21 antibody [EPR362] 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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human papillary carcinoma of the thyroid gland tissue labelling p21 with unpurified Anti-p21 antibody [EPR362] ab109520 at a dilution of 1/100.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling p21 with unpurified Anti-p21 antibody [EPR362] ab109520 at a dilution of 1/100.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Western blot - Anti-p21 antibody [EPR362] - BSA and Azide free (ab218311)
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-p21 antibody [EPR362] ab109520).
Western blot: Anti-CDKN1A antibody [EPR362] (Anti-p21 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, Anti-p21 antibody [EPR362] 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] (Anti-p21 antibody [EPR362] ab109520) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: Western blot - Human CDKN1A knockout HCT116 cell line (Human CDKN1A knockout HCT116 cell line ab288187)
Lane 2: CDKN1A knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 cell lysate at 20 µg
Lane 4: Western blot - Human CDKN1A knockout MCF7 cell line (Human CDKN1A knockout MCF7 cell line ab288200)
Lane 4: CDKN1A knockout MCF7 cell lysate at 20 µg
Lane 5: Wild-type A549 cell lysate at 20 µg
Lane 6: Western blot - Human CDKN1A knockout A549 cell line (Human CDKN1A knockout A549 cell line ab288213)
Lane 6: CDKN1A knockout A549 cell lysate at 20 µg
Secondary
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