Name: Anti-p53 antibody [Y5]
SKU: ab32049
Rating: 4 (5 reviews)

Anti-p53 antibody [Y5] (ab32049) is a rabbit monoclonal antibody detecting p53 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IP, IHC-P, ICC/IF. Suitable for Human.

- KO validated for confirmed specificity

- Biophysical QC for unrivalled batch-batch consistency

- Over 50 publications

- Trusted since 2006

Related conjugates and formulations

ab190335
Conjugated
HRP Anti-p53 antibody [Y5]
ab219731
Carrier free
Anti-p53 antibody [Y5] - BSA and Azide free
ab311682
Conjugated
Alexa Fluor® 594 Anti-p53 antibody [Y5]
ab310851
Conjugated
APC Anti-p53 antibody [Y5]
ab310921
Conjugated
PE Anti-p53 antibody [Y5]
ab311085
Conjugated
Alexa Fluor® 647 Anti-p53 antibody [Y5]
ab224920
Conjugated
Alexa Fluor® 488 Anti- p53 antibody [Y5]
ab321560
Conjugated
Alexa Fluor® 750 Anti-p53 antibody [Y5]

Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (AB32049), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 50% Glycerol (glycerin, glycerine), 49% PBS, 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

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

Abcam Recommends

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 1/50	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/20	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000 - 1/5000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Associated Products

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Target data

See full target information TP53

Function

Multifunctional transcription factor that induces cell cycle arrest, DNA repair or apoptosis upon binding to its target DNA sequence (PubMed:11025664, PubMed:12524540, PubMed:12810724, PubMed:15186775, PubMed:15340061, PubMed:17317671, PubMed:17349958, PubMed:19556538, PubMed:20673990, PubMed:20959462, PubMed:22726440, PubMed:24051492, PubMed:24652652, PubMed:35618207, PubMed:36634798, PubMed:38653238, PubMed:9840937). Acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type (PubMed:11025664, PubMed:12524540, PubMed:12810724, PubMed:15186775, PubMed:15340061, PubMed:17189187, PubMed:17317671, PubMed:17349958, PubMed:19556538, PubMed:20673990, PubMed:20959462, PubMed:22726440, PubMed:24051492, PubMed:24652652, PubMed:38653238, PubMed:9840937). Negatively regulates cell division by controlling expression of a set of genes required for this process (PubMed:11025664, PubMed:12524540, PubMed:12810724, PubMed:15186775, PubMed:15340061, PubMed:17317671, PubMed:17349958, PubMed:19556538, PubMed:20673990, PubMed:20959462, PubMed:22726440, PubMed:24051492, PubMed:24652652, PubMed:9840937). 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 (PubMed:12524540, PubMed:17189187). Its pro-apoptotic activity is activated via its interaction with PPP1R13B/ASPP1 or TP53BP2/ASPP2 (PubMed:12524540). However, this activity is inhibited when the interaction with PPP1R13B/ASPP1 or TP53BP2/ASPP2 is displaced by PPP1R13L/iASPP (PubMed:12524540). 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 seems to have an 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. Regulates the circadian clock by repressing CLOCK-BMAL1-mediated transcriptional activation of PER2 (PubMed:24051492).

Alternative names

P53, TP53, Cellular tumor antigen p53, Antigen NY-CO-13, Phosphoprotein p53, Tumor suppressor p53

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 50% Glycerol (glycerin, glycerine), 49% PBS, 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

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

Clone number

Purification technique

Affinity purification Protein A

Specificity

This antibody clone recognises both wild-type and mutant forms of p53 in human samples. It is not designed to recognise any specific p53 mutation.

We have confirmed this experimentally and have been able to detect p53 in different cell lines using various applications and treatments.

Important note: p53 expression levels vary greatly between cell lines. It has been reported that p53 mutations render the protein more stable, hence mutated cell lines often express higher levels of the p53 protein compared to wild-type cell lines. For low expressing wild type cell lines, p53 expression can be increased with cell treatments such as camptothecin or irinotecan.

Dissociation constant

2.02 x 10^-10 M

Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

What is this antibody validated in?

Anti-p53 antibody [Y5] (ab32049) is a rabbit recombinant monoclonal antibody and is validated for use in Western Blot (WB), Flow Cytometry (Intra), Flow Cytometry (Flow Cyt), Immunoprecipitation (IP), Immunohistochemistry (IHC-P), Immunocytochemistry/immunofluorescence (ICC/IF) in Human samples.

What is the molecular weight of p53?

Anti-p53 [Y5] (ab32049) specifically detects a band for p53 (UniProt: P04637) at a molecular weight of 44kDa.

Trusted by the scientific community

Anti-p53 [Y5] (ab32049) was first used in a scientific publication in 2006 and has been cited over 50 times in peer-reviewed journals.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies.

Specificity confirmed

The specificity of Anti-p53 antibody [Y5] (ab32049) has been confirmed by Western blot testing in TP53 Knockout HAP1 cells.

Other related products

We have a range of other formats of antibody clone [Y5] also available for your convenience:
ab32049, HRP - ab190335, Carrier free - ab219731, Alexa Fluor® 488 - ab224920, APC - ab310851, PE - ab310921, Alexa Fluor® 647 - ab311085, Alexa Fluor® 594 - ab311682, Alexa Fluor® 568 - ab312957, Alexa Fluor® 555 - ab313166, Alexa Fluor® 750 - ab321560

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.

Species reactivity
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species.
Please contact us for more information.

Supplementary info

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

Activity summary

The protein p53 also known as TP53 or tumor protein p53 has a molecular weight of approximately 53 kDa. It acts as a transcription factor and plays a major role in cell cycle regulation apoptosis and maintaining genomic stability. This protein mainly expresses in the nucleus of cells and acts as a critical regulator of cellular responses to stress signals including DNA damage. Scientists commonly use p53 antibodies in various assays like western blot and p53 immunofluorescence to detect and study its expression and functional status in cells.

Biological function summary

P53 functions to control cell division and apoptosis serving as a guardian of the genome by preventing mutation accumulation. It does not form part of a larger complex under normal conditions but interacts with various other molecules to execute its functions. p53 can activate or suppress the transcription of numerous genes involved in cell cycle arrest DNA repair and programmed cell death allowing it to halt the progression of damaged cells and trigger repair mechanisms or eliminate those that cannot be repaired.

Pathways

P53 acts within several key biological pathways such as the p53 signaling pathway and the intrinsic apoptotic pathway. Its activity involves interaction with proteins like MDM2 which regulates p53 through ubiquitin-mediated degradation and ATM kinase which phosphorylates p53 in response to DNA damage. These interactions ensure appropriate cellular responses during stress and are vital for maintaining homeostasis.

Associated diseases and disorders

P53 mutation or inactivation is often associated with the development of cancer given its role in controlling cell division and preventing tumor formation. Specifically its dysfunction has been linked to cancers such as breast cancer and lung cancer. Additionally p53 can interact with other mutant proteins like Ras compounding mutations that contribute to tumor progression and aggressive cancer phenotypes. Understanding these interactions and the status of p53 can be important in developing targeted cancer therapies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
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21 product images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
ab32049 showing positive staining in Urinary bladder carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Western blot - Anti-p53 antibody [Y5] (ab32049)
Blocking/Diluting buffer and concentration: 5% NFDM/TBST
Lane1: Mutant p53 cell lines
Lane2-3: Wildtype p53 cell lines
Exposure time: 180 seconds
Observed MW: 50KDa
All lanes: Western blot - Anti-p53 antibody [Y5] (ab32049) at 1/10000 dilution
Lane 1: A431 (Human epidermoid carcinoma epithelial cell), Whole cell lysate at 20 µg
Lane 2: A549 (Human lung carcinoma epithelial cell), Whole cell lysate at 20 µg
Lane 3: MCF-7 (Human breast adenocarcinoma epithelial cell), Whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 43 kDa
Exposure time: 180s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
Immunohistochemical analysis of paraffin embedded normal Human uterus tissue (negative control) labeling p53 with ab32049.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Western blot - Anti-p53 antibody [Y5] (ab32049)
Blocking/Diluting Buffer and concentration: 5% NFDM/TBST
Lane 1-4: Wildtype p53 cell lines
Lane 5-8: Mutant p53 cell lines
Observed MW: 50 kDa
All lanes: Western blot - Anti-p53 antibody [Y5] (ab32049) at 1/500 dilution
Lane 1: HepG2 (Human hepatocellular carcinoma epithelial cell), Whole cell lysate at 20 µg
Lane 2: A549(Human lung carcinoma epithelial cell), Whole cell lysate at 20 µg
Lane 3: MCF-7(Human breast adenocarcinoma epithelial cell), Whole cell lysate at 20 µg
Lane 4: U-87 MG (Human glioblastoma-astrocytoma epithelial cell), Whole cell lysate at 20 µg
Lane 5: MDA-MB-435(Human mammary gland ductal carcinoma melanocyte), Whole cell lysate at 20 µg
Lane 6: T-47D (Human ductal breast epithelial tumor epithelial cell), Whole cell lysate at 20 µg
Lane 7: Raji (Human Burkitt's lymphoma B lymphocyte), Whole cell lysate at 20 µg
Lane 8: Ramos (Human Burkitt's lymphoma B lymphocyte), Whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 43 kDa
Exposure time: 60s
Flow Cytometry (Intracellular) - Anti-p53 antibody [Y5] (ab32049)
Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling p53 with unpurified ab32049 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
Immunohistochemical analysis of paraffin embedded normal Human breast tissue (negative control) labeling p53 with ab32049.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunoprecipitation - Anti-p53 antibody [Y5] (ab32049)
Mutant p53 was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10 μg with ab32049 at 1/100 dilution (2μg) . VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10 μg
Lane 2: ab32049 IP in A431 whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32049 in A431 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-p53 antibody [Y5] (ab32049)
Predicted band size: 43 kDa
Observed band size: 44 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
Immunohistochemistry (Paraffin-embedded sections) using ab32049 at a dilution of 1/50 and human skin cancer
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
ab32049 showing positive staining in Glioma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
ab32049 showing positive staining in Gastric adenocarcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [Y5] (ab32049)
ab32049 showing positive staining in Breast carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
OI-RD Scanning - Anti-p53 antibody [Y5] (ab32049)
We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.
Flow Cytometry (Intracellular) - Anti-p53 antibody [Y5] (ab32049)
Flow cytometry overlay histogram showing wild-type p53 in Hek-293 positive cells (left) and MCF7 negative cells (right) stained with ab32049 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32049) (1x 106 cells in 100μl at 0.008μg/ml (1/13250)) for 30min at 22°C. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in Hek-293 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
Western blot - Anti-p53 antibody [Y5] (ab32049)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-p53 antibody [Y5] (ab32049) staining at 1/1000 dilution.
All lanes: Western blot - Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] (Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] ab322465) at 1/1000 dilution
Lane 1: 293T (human embryonic kidney epithelial cell) whole cell lysate (untreated membrane) at 20 µg
Lane 2: 293T whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 53 kDa, 35-48 kDa, 36 kDa
Exposure time: 10s
Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [Y5] (ab32049)
ab32049 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at °C with ab32049 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
Also suitable in cells fixed with 100% methanol (5 min).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Flow Cytometry (Intracellular) - Anti-p53 antibody [Y5] (ab32049)
Flow cytometry overlay histogram showing p53 in wild-type HAP1 (green line) and TP53 knockout HAP1 (red line) cells stained with ab32049. The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32049) (1x 106 cells in 100μl at 0.2 μg/ml (1/440)) for 30min at 22°C. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) was used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line, TP53 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in HAP1 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
Western blot - Anti-p53 antibody [Y5] (ab32049)
False colour image of Western blot: Anti-p53 antibody [Y5] 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, ab32049 was shown to bind specifically to p53. A band was observed at 50 kDa in wild-type HAP1 cell lysate with no signal observed at this size in tp53 knockout cell line. To generate this image, wild-type and tp53 knockout HAP1 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-p53 antibody [Y5] (ab32049) at 1/1000 dilution
Lane 1: Saos-2 cell lysate at 20 µg
Lane 2: A431 cell lysate at 20 µg
Lane 3: Wild-type HAP1 cell lysate at 20 µg
Lane 4: TP53 knockout HAP1 cell lysate at 20 µg
Lane 5: MCF7 cell lysate at 20 µg
Lane 6: HEK-293T cell lysate at 20 µg
Secondary
Lanes 1 - 6: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution
Lanes 1 - 6: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 43 kDa
Observed band size: 50 kDa
Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [Y5] (ab32049)
ab32049 staining wild-type p53 in MCF7 cells (a low expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab32049 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [Y5] (ab32049)
ab32049 staining wild-type p53 in Hek293 cells (a high expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab32049 at 0.2µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [Y5] (ab32049)
ab32049 staining mutant p53 in A431 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab32049 at 0.2µ?g/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Western blot - Anti-p53 antibody [Y5] (ab32049)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
In Western blot, Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] ab322465 was shown to bind specifically to TP53. Target of interest was observed at 35-53 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in TP53 knockout HAP1 cell line (lane 2).
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-p53 antibody [Y5] (ab32049) staining at 1/1000 dilution.
All lanes: Western blot - Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] (Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] ab322465) at 1/1000 dilution
Lane 1: Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg
Lane 2: TP53 knockout HAP1 whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 53 kDa, 35-48 kDa, 36 kDa
Exposure time: 15s