Abcam is committed to meeting high standards of ethical manufacturing and has decided to discontinue this product by June 2019 as it has been generated by the ascites method. We are sorry for any inconvenience this may cause. We would recommend antibody ab109520 as a replacement.
Shipped at 4°C. Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 2 µg/ml. Predicted molecular weight: 18 kDa.
Use at 2 µg/mg of lysate. Native verified. Use Protein A.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use 1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
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.
Expressed in all adult human tissues, with 5-fold lower levels observed in the brain.
Belongs to the CDI family.
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.
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.
Melanoma differentiation-associated protein 6 antibody
Melanoma differentiation-associated protein antibody
P21 protein antibody
Wild type p53 activated fragment 1 (WAF1) antibody
Wild type p53 activated fragment 1 antibody
Wildtype p53-activated fragment 1 antibody
Western blot - Anti-p21 antibody [CP74] (ab80633)
Lane 1: Wild type DLD-1 whole cell lysate (20 µg) Lane 2: DLD-1 2,3 DCPE treated whole cell lysate (20 µg) Lane 3: DLD-1 p21 (KO) control whole cell lysate (20 µg) Lane 4: DLD-1 p21(KO) 2,3 DCPE treated whole cell lysate (20 µg) Lane 5: HT1080 whole cell lysate (20 µg) Lanes 1 - 5: Merged signal (red and green). Green - ab80633 observed at 21 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab80633 was shown to specifically react with p21 in DLD-1 cells in the presence of 2,3 DCPE treatment. No band was observed in knockout samples +/-2,3 DCPE treatment. Wild-type and DLD-1 2,3 DCPE treated knockout samples were subjected to SDS-PAGE. Ab80633 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/30000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
IHC image of ab80633 staining in Breast Cancer formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab80633, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-p21 antibody [CP74] (ab80633)Image from Linley AJ et al, J Biol Chem. 2012 Apr 20;287(17):13633-43. Epub 2012 Mar 5, Fig 3. DOI 10.1074/jbc.M111.308973 April 20, 2012 The Journal of Biological Chemistry, 287, 13633-13643.
ab80633 used at a 1/250 dilution for Western Blot. Human cancer cells were collected, washed with 1× PBS, lysed in 1× solution containing 50 mm Tris-HCl (pH 6.8), 100 mm dithiothreitol, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, and 10% (v/v) glycerol and loaded on Tris/glycine SDS-polyacrylamide gels. Proteins were separated alongside a molecular weight marker. Protein bands were transferred onto PVDF membranes. Membranes were blocked with 10% milk/TBS solution with 0.05% Tween 20 containing sodium orthovanadate and sodium fluoride. Following TBS solution with 0.05% Tween 20 washes, membranes were incubated with primary antibodies (in blocking solution) at 4 °C overnight followed by washing and incubation with secondary antibodies for 1 hour at room temperature.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [CP74] (ab80633)This image is courtesy of an anonymous Abreview
Ab80633 staining p21 in human endometriosis tissue sections by Immunohistochemistry. Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. Samples were fixed with formaldehyde and blocked with 3% serum for 30 minutes at 20°C. Samples were incubated with primary antibody at 1/200 dilution for 12 hours at 4°C. An Alexa Fluor® 488 goat anti mouse was used as the secondary antibody at 1/100 dilution.
Overlay histogram showing HeLa cells stained with ab80633 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween 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 (ab80633, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was anti-mouse DyLight® 488 (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.