Recombinant Anti-Mutant p53 antibody [E47] (ab32509)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E47] to Mutant p53
- Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, WB, IP
- Reacts with: Human
Related conjugates and formulations
Overview
-
Product name
Anti-Mutant p53 antibody [E47]
See all Mutant p53 primary antibodies -
Description
Rabbit monoclonal [E47] to Mutant p53 -
Host species
Rabbit -
Specificity
ab32509 recognises human mutant forms of p53 but not human p53 wild type. ab32059 showed a negative signal on wildtype p53 cell lines (HepG2, A549, MCF-7) and a positive signal on mutant p53 cell lines (T47-D, Raji, A431) in WB. -
Tested applications
Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, WB, IPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide within Human Mutant p53 aa 350 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: P04637 -
Positive control
- WB: A431 and T-47D cell lysate ICC/IF: A431 cells. IHC-P: Human lung carcinoma tissue. Flow Cyt (intra): HEK-293 cells IP: HEK-293 cell lysates
-
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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Properties
-
Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
-
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E47 -
Isotype
IgG -
Research areas
Associated products
-
Alternative Versions
-
Isotype control
-
Positive Controls
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32509 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 |
---|---|---|
Flow Cyt (Intra) |
1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
|
IHC-P |
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
|
|
ICC/IF | (1) |
1/100 - 1/250.
|
WB |
1/1000 - 1/10000. Detects a band of approximately 53 kDa (predicted molecular weight: 44 kDa).
|
|
IP |
1/50.
|
Notes |
---|
Flow Cyt (Intra)
1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IHC-P
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/100 - 1/250. |
WB
1/1000 - 1/10000. Detects a band of approximately 53 kDa (predicted molecular weight: 44 kDa). |
IP
1/50. |
Target
-
Function
Acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. Involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. Apoptosis induction seems to be mediated either by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. In cooperation with mitochondrial PPIF is involved in activating oxidative stress-induced necrosis; the function is largely independent of transcription. Induces the transcription of long intergenic non-coding RNA p21 (lincRNA-p21) and lincRNA-Mkln1. LincRNA-p21 participates in TP53-dependent transcriptional repression leading to apoptosis and seem to have to effect on cell-cycle regulation. Implicated in Notch signaling cross-over. Prevents CDK7 kinase activity when associated to CAK complex in response to DNA damage, thus stopping cell cycle progression. Isoform 2 enhances the transactivation activity of isoform 1 from some but not all TP53-inducible promoters. Isoform 4 suppresses transactivation activity and impairs growth suppression mediated by isoform 1. Isoform 7 inhibits isoform 1-mediated apoptosis. -
Tissue specificity
Ubiquitous. Isoforms are expressed in a wide range of normal tissues but in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues but is not detected in brain, lung, prostate, muscle, fetal brain, spinal cord and fetal liver. Isoform 3 is expressed in most normal tissues but is not detected in lung, spleen, testis, fetal brain, spinal cord and fetal liver. Isoform 7 is expressed in most normal tissues but is not detected in prostate, uterus, skeletal muscle and breast. Isoform 8 is detected only in colon, bone marrow, testis, fetal brain and intestine. Isoform 9 is expressed in most normal tissues but is not detected in brain, heart, lung, fetal liver, salivary gland, breast or intestine. -
Involvement in disease
TP53 is found in increased amounts in a wide variety of transformed cells. TP53 is frequently mutated or inactivated in about 60% of cancers. TP53 defects are found in Barrett metaplasia a condition in which the normally stratified squamous epithelium of the lower esophagus is replaced by a metaplastic columnar epithelium. The condition develops as a complication in approximately 10% of patients with chronic gastroesophageal reflux disease and predisposes to the development of esophageal adenocarcinoma.
Esophageal cancer
Li-Fraumeni syndrome
Squamous cell carcinoma of the head and neck
Lung cancer
Choroid plexus papilloma
Adrenocortical carcinoma
Basal cell carcinoma 7 -
Sequence similarities
Belongs to the p53 family. -
Domain
The nuclear export signal acts as a transcriptional repression domain. The TADI and TADII motifs (residues 17 to 25 and 48 to 56) correspond both to 9aaTAD motifs which are transactivation domains present in a large number of yeast and animal transcription factors. -
Post-translational
modificationsAcetylated. Acetylation of Lys-382 by CREBBP enhances transcriptional activity. Deacetylation of Lys-382 by SIRT1 impairs its ability to induce proapoptotic program and modulate cell senescence.
Phosphorylation on Ser residues mediates transcriptional activation. Phosphorylated by HIPK1 (By similarity). Phosphorylation at Ser-9 by HIPK4 increases repression activity on BIRC5 promoter. Phosphorylated on Thr-18 by VRK1. Phosphorylated on Ser-20 by CHEK2 in response to DNA damage, which prevents ubiquitination by MDM2. Phosphorylated on Ser-20 by PLK3 in response to reactive oxygen species (ROS), promoting p53/TP53-mediated apoptosis. Phosphorylated on Thr-55 by TAF1, which promotes MDM2-mediated degradation. Phosphorylated on Ser-33 by CDK7 in a CAK complex in response to DNA damage. Phosphorylated on Ser-46 by HIPK2 upon UV irradiation. Phosphorylation on Ser-46 is required for acetylation by CREBBP. Phosphorylated on Ser-392 following UV but not gamma irradiation. Phosphorylated on Ser-15 upon ultraviolet irradiation; which is enhanced by interaction with BANP. Phosphorylated by NUAK1 at Ser-15 and Ser-392; was intially thought to be mediated by STK11/LKB1 but it was later shown that it is indirect and that STK11/LKB1-dependent phosphorylation is probably mediated by downstream NUAK1 (PubMed:21317932). It is unclear whether AMP directly mediates phosphorylation at Ser-15. Phosphorylated on Thr-18 by isoform 1 and isoform 2 of VRK2. Phosphorylation on Thr-18 by isoform 2 of VRK2 results in a reduction in ubiquitination by MDM2 and an increase in acetylation by EP300. Stabilized by CDK5-mediated phosphorylation in response to genotoxic and oxidative stresses at Ser-15, Ser-33 and Ser-46, leading to accumulation of p53/TP53, particularly in the nucleus, thus inducing the transactivation of p53/TP53 target genes. Phosphorylated by DYRK2 at Ser-46 in response to genotoxic stress. Phosphorylated at Ser-315 and Ser-392 by CDK2 in response to DNA-damage.
Dephosphorylated by PP2A-PPP2R5C holoenzyme at Thr-55. SV40 small T antigen inhibits the dephosphorylation by the AC form of PP2A.
May be O-glycosylated in the C-terminal basic region. Studied in EB-1 cell line.
Ubiquitinated by MDM2 and SYVN1, which leads to proteasomal degradation. Ubiquitinated by RFWD3, which works in cooperation with MDM2 and may catalyze the formation of short polyubiquitin chains on p53/TP53 that are not targeted to the proteasome. Ubiquitinated by MKRN1 at Lys-291 and Lys-292, which leads to proteasomal degradation. Deubiquitinated by USP10, leading to its stabilization. Ubiquitinated by TRIM24, which leads to proteasomal degradation. Ubiquitination by TOPORS induces degradation. Deubiquitination by USP7, leading to stabilization. Isoform 4 is monoubiquitinated in an MDM2-independent manner.
Monomethylated at Lys-372 by SETD7, leading to stabilization and increased transcriptional activation. Monomethylated at Lys-370 by SMYD2, leading to decreased DNA-binding activity and subsequent transcriptional regulation activity. Lys-372 monomethylation prevents interaction with SMYD2 and subsequent monomethylation at Lys-370. Dimethylated at Lys-373 by EHMT1 and EHMT2. Monomethylated at Lys-382 by SETD8, promoting interaction with L3MBTL1 and leading to repress transcriptional activity. Dimethylation at Lys-370 and Lys-382 diminishes p53 ubiquitination, through stabilizing association with the methyl reader PHF20. Demethylation of dimethylated Lys-370 by KDM1A prevents interaction with TP53BP1 and represses TP53-mediated transcriptional activation.
Sumoylated with SUMO1. Sumoylated at Lys-386 by UBC9. -
Cellular localization
Cytoplasm; Nucleus. Cytoplasm. Localized in both nucleus and cytoplasm in most cells. In some cells, forms foci in the nucleus that are different from nucleoli; Nucleus. Cytoplasm. Localized in the nucleus in most cells but found in the cytoplasm in some cells; Nucleus. Cytoplasm. Localized mainly in the nucleus with minor staining in the cytoplasm; Nucleus. Cytoplasm. Predominantly nuclear but localizes to the cytoplasm when expressed with isoform 4; Nucleus. Cytoplasm. Predominantly nuclear but translocates to the cytoplasm following cell stress and Cytoplasm. Nucleus. Nucleus > PML body. Endoplasmic reticulum. Mitochondrion matrix. Interaction with BANP promotes nuclear localization. Recruited into PML bodies together with CHEK2. Translocates to mitochondria upon oxidative stress. - Information by UniProt
-
Database links
- Entrez Gene: 7157 Human
- Omim: 191170 Human
- SwissProt: P04637 Human
- Unigene: 437460 Human
- Unigene: 740601 Human
-
Alternative names
- Antigen NY-CO-13 antibody
- BCC7 antibody
- Cellular tumor antigen p53 antibody
see all
Images
-
All lanes : Anti-Mutant p53 antibody [E47] (ab32509) at 1/200 dilution
Lane 1 : A549 (Human lung carcinoma epithelial cell), Whole cell lysate
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell), Whole cell lysate
Lane 3 : MCF-7 (Human breast adenocarcinoma epithelial cell), Whole cell lysate
Lane 4 : T-47D (Human ductal breast epithelial tumor epithelial cell), Whole cell lysate
Lane 5 : A431 (Human epidermoid carcinoma epithelial cell), Whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 44 kDaBlocking/Diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 180 seconds
Lane 1-3: Wildtype p53 cell lines
Lane 4-5: Mutant p53 cell lines
Observed MW: 50kDa, 39kDa
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mutant p53 antibody [E47] (ab32509)Immunohistochemical analysis of paraffin-embedded human lung carcinoma sections labeling mutant p53 with ab32509 at 1/2000 dilution (0.49 μg/mL). Sections were counterstained with Hematoxylin. Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).Nuclear staining on human lung carcinoma.
-
Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) labelling mutant p53 with purified ab32509 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).
Control: PBS only.
-
Purified ab32509 at 1/50 dilution (2µg) immunoprecipitating Mutant p53 in HEK-293 whole cell lysate.
Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32509 + HEK-293 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32509 in HEK-293 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 53 kDa -
Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling Mutant p53 with purified ab32509 at 1/100 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).
-
Anti-Mutant p53 antibody [E47] (ab32509) at 1/1000 dilution + A431 lysate
Predicted band size: 44 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
-
Datasheet download
References (11)
ab32509 has been referenced in 11 publications.
- Li YP et al. Piperine protects against myocardial ischemia/reperfusion injury by activating the PI3K/AKT signaling pathway. Exp Ther Med 21:374 (2021). PubMed: 33732347
- Kishi S et al. Role of Glycated High Mobility Group Box-1 in Gastric Cancer. Int J Mol Sci 22:N/A (2021). PubMed: 34068442
- Huang J et al. Combined effects of low-dose gambogic acid and NaI131 in drug-resistant non-small cell lung cancer cells. Oncol Lett 22:588 (2021). PubMed: 34149899
- Huo L et al. Leukemia Inhibitory Factor Receptor Is Involved in Apoptosis in Rat Astrocytes Exposed to Oxygen-Glucose Deprivation. Biomed Res Int 2019:1613820 (2019). PubMed: 30937308
- Zhang BF et al. Nobiletin ameliorates myocardial ischemia and reperfusion injury by attenuating endoplasmic reticulum stress-associated apoptosis through regulation of the PI3K/AKT signal pathway. Int Immunopharmacol 73:98-107 (2019). PubMed: 31082728