Anti p53

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, France, Germany

Anti-p53 is a laboratory reagent used to detect the presence and localization of the p53 protein in biological samples. p53 is a tumor suppressor protein that plays a critical role in cellular processes such as cell cycle regulation, apoptosis, and DNA repair. Anti-p53 can be used in various techniques, including Western blotting, immunohistochemistry, and immunofluorescence, to study the expression and distribution of p53 in cells and tissues.

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Anti-Phospho-p53 (Ser15) Antibody Mouse anti-p53 Rabbit anti-p53 pS15 Rabbit anti-p53 antibody Rabbit anti-p53 Anti-p53-S15 Anti-p53 (clone 1C12) Mouse anti-human p53 Anti-p53 monoclonal antibody Anti–acetylated p53

256 protocols using anti p53

Comprehensive Antibody Panel for Cell Signaling Analysis

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Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.

Yang L., Li Y., Bhattacharya A, & Zhang Y. (2017). PEPD is a pivotal regulator of p53 tumor suppressor. Nature Communications, 8, 2052.

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Nutlin-3a Modulates p53-MDM2 Interactions

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Exponentially growing PA-1 cells were treated with 40 μM nutlin-3a and equivalent volume of DMSO. Whole-cell extracts were generated using lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40]. Protein extracts (500 μg) were precleared for 2 h with 40 μL protein G Sepharose beads (50%, Sigma) before addition of the indicated antibodies. For immunoprecipitation, rabbit monoclonal antibody anti-p53 (Cell Signaling Technology, 2 mg/mL) and mouse monoclonal anti-MDM2 (abcam, 1 mg/mL) were used. Immune complexes were then collected on protein G Sepharose beads at 4°C overnight, and beads were washed five times with cold lysis buffer. Precipitated proteins were subjected to Western blotting with rabbit monoclonal antibody anti-p53 (Cell Signaling Technology), mouse monoclonal anti-MDM2(abcam, 1 mg/mL), polyclonal antibodies pan-actin (Cell Signaling Technology, 2 mg/mL).

Hu K., Yin F., Yu M., Sun C., Li J., Liang Y., Li W., Xie M., Lao Y., Liang W, & Li Z.G. (2017). In-Tether Chiral Center Induced Helical Peptide Modulators Target p53-MDM2/MDMX and Inhibit Tumor Growth in Stem-Like Cancer Cell. Theranostics, 7(18), 4566-4576.

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Investigating DNA Damage and Apoptosis

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Activation of p53 induces the expression of various gene products, which in turn can either prevent proliferation of damaged cells or induce apoptosis, thereby removing damaged cells from the body. 22 Phosphorylation of H2AX plays a major role in the DNA damage response and is required for the assembly of DNA repair proteins. 23 Therefore, we investigated these molecules, given their role in apoptosis and DNA damage. 9, 23 For analysis of the xenograft model, selected tumors excised from the treated and control mice were stained for anti-pH2AX and anti-p53 (Cell Signaling Technology). To further investigate the effect of EDO-S101 on DNA damage, rat spleen sections (10 mm) were also stained for anti-pH2AX and anti-p53 (Cell Signaling Technology). For analysis of the vasculitis model, kidney sections (4 mm) were stained with periodic acid-Schiff and hematoxylin and eosin stains to assess morphology as previously described. [19] [20] [21] 24 In addition to renal pathology, rats with EAV develop pulmonary vasculitis and hemorrhage as previously described 25 (see Supplementary Methods for further details).

Dooley D., van Timmeren M.M., O'Reilly V.P., Brady G., O'Brien E.C., Fazekas B., Hickey F.B., Leacy E., Pusey C.D., Tam F.W.K., Mehrling T., Heeringa P, & Little M.A. (2018). Alkylating histone deacetylase inhibitors may have therapeutic value in experimental myeloperoxidase-ANCA vasculitis. Kidney international, 94(5).

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Protein Interaction Analysis by Co-IP

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Preparation of cell extracts, co-immunoprecipitation and immunoblotting were carried out as previously described (Pyronnet et al., 1999 (link)). The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).

Alard A., Marboeuf C., Fabre B., Jean C., Martineau Y., Lopez F., Vende P., Poncet D., Schneider R.J., Bousquet C, & Pyronnet S. (2019). Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome. Frontiers in Genetics, 10, 254.

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Comprehensive Protein Analysis Protocol

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The following Abs were used in this study: anti‐Girdin (R&D Systems), anti‐Girdin (IBL), anti‐Girdin phospho S1647 (ECM Biosciences), anti‐histone H3 (1B1B2) (Cell Signaling Technology), anti‐histone H3 phospho S10 (Abcam), anti‐histone H3 phospho S28 (Abcam), anti‐cleaved PARP1 (Abcam), anti‐cleaved PARP1 (Cell Signaling Technology), anti‐Rb phospho Ser795 (New England BioLabs), anti‐Rb (4H1) (Cell Signaling Technology), anti‐p53 (Cell Signaling Technology), anti‐p53 phospho S15 (Cell Signaling Technology), anti‐p53 phospho S46 (Cell Signaling Technology), anti‐Mad2 (C‐10) (Santa Cruz Biotechnology), anti‐α‐tubulin (Sigma‐Aldrich), anti‐γ‐tubulin (Sigma‐Aldrich), Alexa Fluor 488 goat anti‐mouse IgG (Thermo Fisher Scientific), Alexa Fluor 488 goat anti‐rabbit IgG (Thermo Fisher Scientific), rabbit anti‐sheep IgG (H + L), Human SP ads‐HRP (Southern Biotech), and rabbit anti‐rat IgG H&L (HRP) (Abcam) Abs.

Chen C., Enomoto A., Weng L., Taki T., Shiraki Y., Mii S., Ichihara R., Kanda M., Koike M., Kodera Y, & Takahashi M. (2020). Complex roles of the actin‐binding protein Girdin/GIV in DNA damage‐induced apoptosis of cancer cells. Cancer Science, 111(11), 4303-4317.

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Western Blot Analysis of Cell Signaling

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Western blot analysis was performed according to a standard protocol.⁴⁴ (link) The primary antibodies included anti-ACP5 (Gentex, CA, USA, 1:1,000), anti-fibronectin, anti-E-cadherin, anti-vimentin (Proteintech, Wuhan, China, 1:1,000), anti-p38, anti-ERK, anti-p-ERK, anti-AKT, anti-β-catenin, anti-SMAD2/3, anti-p-SMAD2, anti-p-SMAD3, anti-p53, anti-p53 (Ser392), anti-ubiquitin (Cell Signaling Technology, Danvers, MA, USA, 1:1,000), anti-GAPDH, and anti-β-actin (Abcam, Cambridge, MA, USA, 1:3,000) antibodies. Detection was performed using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). The gray values were analyzed with ImageJ software.

Hu Y., Yu J., Wang Q., Zhang L., Chen X., Cao Y., Zhao J., Xu Y., Jiang D., Wang Y, & Xiong W. (2020). Tartrate-Resistant Acid Phosphatase 5/ACP5 Interacts with p53 to Control the Expression of SMAD3 in Lung Adenocarcinoma. Molecular Therapy Oncolytics, 16, 272-288.

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Western Blot Analysis of Apoptosis Markers

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Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies (Grand Island, NY, USA). The anti-Bak, anti-PARP, anti-Bcl-2, anti-p53, anti-phospho-p53, anti-acetyl-p53, anti-SIRT1, and anti-cyclin D1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-b-actin antibody was from Millipore Corp. (Temecula, CA, USA). The antisera to tNOX used in Western blot analyses were generated as described previously [22 (link)]. The 3,3′-Dihexyloxacarbocyanine iodide [DiOC6(3)] was purchased from Calbiochem (San Diego, CA, USA). The anti-mouse and anti-rabbit IgG antibodies and other chemicals were purchased from the Sigma Chemical Company (St. Louis, MO, USA), unless specified otherwise.

Chen H.Y., Cheng H.L., Lee Y.H., Yuan T.M., Chen S.W., Lin Y.Y, & Chueh P.J. (2017). Tumor-associated NADH oxidase (tNOX)-NAD+-sirtuin 1 axis contributes to oxaliplatin-induced apoptosis of gastric cancer cells. Oncotarget, 8(9), 15338-15348.

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SINE Compounds Inhibit Nuclear Export

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The SINE compounds KPT-185 and selinexor (KPT-330, molecular weight 443.31, chemical formula C₁₇H₁₁F₆N₇O) were obtained from Karyopharm Therapeutics Inc. (Natick, MA). KPT-185 is suitable for in vitro use and selinexor is suitable for in vivo use. Primary antibodies used included anti-p53 (Cell Signaling, Danvers, MA), anti-lamin B1 (Life Technologies, Grand Islands, NY), anti-tubulin (Cell Signaling), anti-COX IV (Abcam, Cambridge, MA), anti-beta actin (Sigma-Aldrich, St. Louis, MO), anti-eIF5A (Abcam), anti-IGF2BP1 (Abcam), anti-Ki67 (Thermo Lab Vision, Kalamazoo, MI), and anti-cleaved caspase 3 (Cell Signaling). The following secondary antibodies were used: horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G, horseradish peroxidase–conjugated rat anti-mouse immunoglobulin G2a (Serotec Harlan Bioproducts for Science, Inc., Indianapolis, IN), and fluorescent Alexa 594 immunoglobulin G (Life Technologies).

Miyake T., Pradeep S., Wu S.Y., Rupaimoole R., Zand B., Wen Y., Gharpure K.M., Nagaraja A.S., Hu W., Cho M.S., Dalton H.J., Previs R.A., Taylor M.L., Hisamatsu T., Kang Y., Liu T., Shacham S., McCauley D., Hawke D.H., Wiktorowicz J.E., Coleman R.L, & Sood A.K. (2015). XPO1/CRM1 Inhibition Causes Antitumor Effects by Mitochondrial Accumulation of eIF5A. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(14), 3286-3297.

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Comprehensive Protein Analysis Protocol

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IU1, siRNA and shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MG132 and bortezomib (Velcade) were purchased from Selleckchem (Houston, TX, USA). MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). Propidium iodide (PI) and Annexin V-FITC Apoptosis Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies. Antibodies used in this study were purchased from following sources: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology); anti-PARP, anti-CDK2, anti-phospho-Rb, anti-Rb, anti-PSA, anti-Bax, anti-GFP, anti-GAPDH (Bioworld Technology, Inc., Louis Park, MN, USA); anti-CDK4, anti-CDK6, anti-phospho-MDM2, anti-P53, anti-USP14, anti-Flag, anti-cyclin D1, anti-p15, anti-p27 (Cell Signaling Technology, Beverly, MA, USA); anti-MDM2 and anti-AR (Abcam, USA).

Liao Y., Liu N., Hua X., Cai J., Xia X., Wang X., Huang H, & Liu J. (2017). Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor. Cell Death & Disease, 8(2), e2585-.

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Antibody Immunoblotting Panel

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Rabbit anti-NCKAP1 was purchased from Proteintech. Rabbit anti-WASF1, anti-pRb, anti-P53, anti-cyclin D1, anti-p27, anti-CDK2, and anti-CDK4 antibodies and mouse anti-p18 and anti-CDK6 antibodies were purchased from Cell Signaling Technology.

Zhong X.P., Kan A., Ling Y.H., Lu L.H., Mei J., Wei W., Li S.H, & Guo R.P. (2019). NCKAP1 improves patient outcome and inhibits cell growth by enhancing Rb1/p53 activation in hepatocellular carcinoma. Cell Death & Disease, 10(5), 369.

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