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.
FunctionMay 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 specificityExpressed in all adult human tissues, with 5-fold lower levels observed in the brain.
Sequence similaritiesBelongs to the CDI family.
DomainThe 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.
Lane 1: Wild type DLD-1 whole cell lysate (20 µg) Lane 2: DLD-1 2,3 DCPE treated knockout DLD-1 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 recognize p21 when DLD-1 2,3 DCPE treated knockout samples were used, along with additional cross-reactive bands. 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] - BSA and Azide free (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.
Predicted band size : 18 kDa
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.
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.
References for Anti-p21 antibody [CP74] (ab80633)
This product has been referenced in:
Kilarkaje N & Al-Bader MM Diabetes-Induced Oxidative DNA Damage Alters p53-p21CIP1/Waf1 Signaling in the Rat Testis. Reprod SciN/A:N/A (2014).
Read more (PubMed: 24828139) »
Linley AJ et al. The Helicase HAGE Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Cell Proliferation in Vivo. J Biol Chem287:13633-43 (2012).
Read more (PubMed: 22393060) »