This is a knockout-validated antibody summary, based on publications "A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3" [7] (western blot, Figure s5b in the article), "Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma" [8] (western blot, see figure 2a in the article), Dependence of p53-deficient cells on the DHX9 DExH-box helicase" [9] (western blot, figure 1f), "STIP is a critical nuclear scaffolding protein linking USP7 to p53-Mdm2 pathway regulation" [1], "Loss of PTEN Facilitates Rosiglitazone-Mediated Enhancement of Platinum(IV) Complex LA-12-Induced Apoptosis in Colon Cancer Cells" [2], "The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells" [3], "High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis" [4], "Sequential cancer mutations in cultured human intestinal stem cells" [10], "Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance" [5], "A chemical genetics approach for the functional assessment of novel cancer genes" [11], "Nerve growth factor receptor negates the tumor suppressor p53 as a feedback regulator" [6], "Pleckstrin homology domain-containing protein PHLDB3 supports cancer growth via a negative feedback loop involving p53" [12] (Figure 1b), "Acetylation-regulated interaction between p53 and SET reveals a widespread regulatory mode" [13], "Development of a genetic sensor that eliminates p53 deficient cells" (immunocytochemistry and western blot, figures 2b and 2c) [14], "p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells" (western blot, figure 2d) [15], "p53-dependent autophagic degradation of TET2 modulates cancer therapeutic resistance" (see figure 1b, 4c in the article) [16], "RNA-binding motif protein 10 induces apoptosis and suppresses proliferation by activating p53" for western blot knockout validation (figure 1b) [17], "Targeting a neoantigen derived from a common TP53 mutation" for western blot knockout validation (figure s6a) [18], "Long noncoding RNA AGPG regulates PFKFB3-mediated tumor glycolytic reprogramming" for western blot knockout validation (figure 5b) [19] "CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer" for western blot knockout validation (figure 1f) [20], "DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance" for western blot knockout validation (figure 2a) [21], "Contribution of p53 in sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer" for western blot knockout validation (figure 1a) [22], and "TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling" for western blot knockout validation (figure 1b) [23]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Mouse monoclonal IgG2a (kappa light chain)

Company: Santa Cruz

Antibody: p53

Catalog number: sc-126

Clone: DO-1. This clone is also sold as Bio-Rad MCA1701B, MCA1701F, MCA1701PE, MCA1701; BioLegend 645702, 645701; LifeSpan Biosciences LS-C11553, LS-C131180, LS-C87895, LS-C87898, LS-C179709; US Biological P1001-26A5, P1001-26R, P1001-26A1, P1001-32C, P1001-32N, P1001-26U1; Abcam ab80645; Active Motif 39553; Invitrogen MA5-12571, AHO0152; MyBioSource MBS212262; Santa Cruz Biotechnology sc-126, sc-126 HRP, sc-126 X, sc-126 AC.

Summary: Mouse monoclonal IgG2a (kappa light chain) against an N-terminal epitope mapping between amino acid residues 11-25 of p53 of human origin. Recommended for detection of wild type and mutant p53 under denaturing and non-denaturing conditions of human origin by western blot, immunoprecipitation, immunofluorescence, immunohistochemistry (paraffin) and flow cytometry.

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Figure 1. Western blot of p53 in U2OS (p53+/+), H1299 (p53−/−), HCT116 (p53+/+), and HCT116 (p53−/−) cells. Actin served as loading control. From [1].

Western blot (Figure 1).

U2OS (p53+/+), H1299 (p53−/−), HCT116 (p53+/+), and HCT116 (p53−/−) cells. Whole cell lysates were prepared with M-PER buffer containing protease inhibitors (Pierce).

5% nonfat milk.

overnight at 4°C.

horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).

ECL Kit (Pierce).

Authors do not specify which of the two antibodies (another one is Bethyl A300-247A) was used for the western blot. We assume both are KO validated.

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Figure 2. Western blotting of p53 protein levels in HCT116 wt and p53-/- cells. β-actin served as loading control. From [2].

Western blot (Figure 2).

Human colon adenocarcinoma cell lines HCT116 wt (p53+/+, Bax+/-, Chk2+/+, PTEN+/+) and p53-/-. Cells were lysed in 1% SDS lysis buffer containing protease inhibitor cocktail (#P2714, Sigma–Aldrich) or Protease Inhibitor Cocktail Set I (#5391313, Calbiochem, Merck Millipore, Bedford, MA, USA), for phosphoprotein detection NaVO4 and NaF were added to the lysis solution.

5% non-fat milk or BSA for 1 h at RT.

1:1,000 dilution in 5% non-fat milk at 4°C overnight.

anti-mouse IgG (1:3,000, #NA931) (Amersham Biosciences, Piscataway, NJ, USA) for 1 h at RT.

Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0500, Merck Millipore, Bedford, MA, USA).

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Figure 3. From Figure 5 in [3]. Western blot of lysates of human non-small cell lung cancer cell lines (A549, p53 positive; H1299, p53 null) probed with anti-p53 antibody. Actin serves as a loading control.

Western blot (Figure 3).

Lysates of human non-small cell lung cancer cell lines (A549, p53 positive; H1299, p53 null).

Antip53 antibody.

Chemiluminescence.

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Figure 4. The genome of HCT116 cells was targeted using sgATRX-E4 and sgTP53-E4.2. Western blot analyses with sc-126 were performed on two double knockout clones (ATRX-/- TP53-/-) are. From Figure 6d in [4].

Western blot (Figure 4).

Confluent HCT116 human colorectal carcinoma cells were lysed with Lysis 250 Buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 5 mM EDTA pH 8.0, 0.1% NP-40, 50 mM NaF) supplemented with Complete Protease Inhibitor (Hoffmann-La Roche), by a series of freeze-thaw cycles.

anti-p53 antibody was diluted 1:1,000. The portion of article indicates an anti-p53 antibody from Abcam; however, no Abcam antibody was listed in the text section about the sources of antibodies. Therefore it is likely a typo.

Sheep anti-mouse HRP-conjugated antibody (1:20,000; Jackson ImmunoResearch).

Western blot

Wildtype and P53KO human organoids. Samples were lysed using RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Na-Deoxycholate, 1% NP-40) containing Complete protease inhibitors (Roche).

Please see Figure 2f in the article [10].

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Figure 5. Western blot of p53 and α-Tubulin (loading control) in WT p53+/+ and p53-/- HCT116 cells. From [5].

Western blot (Figure 5).

WT p53+/+ and p53-/- HCT116 cells. cells were collected in lysis buffer (25 mM HEPES pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 20 mM β-glycerophosphate) plus protease and phosphatase inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF and 0.1 mM Na3VO4).

Western blot

WT and DD-p53 tagged (by TALEN knockin) HCT116 cells.

Goat anti-mouse IgG-HRP (sc-2031; Santa Cruz Biotechnology)

Please see Figure 1b in the article [11].

This is not a classical KO validation, but could be considered one. The protein is KI tagged and there is a clear shift in the western blot, while no original protein is detected.

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Figure 6. Immunoblot of p53 and β-actin (loading control) in HCT116 p53+/+ and HCT116 p53-/- cells treated with Doxorubicin or 5-Fluorouracil. From [6].

Western blot (Figure 6).

HCT116 p53+/+ and HCT116 p53-/- cells. Cells were harvested and lysed in lysis buffer consisting of 50 mM Tris/HCl (pH7.5), 0.5% Nonidet P-40 (NP-40), 1 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 µM pepstatin A and 1 mM leupeptin.

The same clone (DO-1) is sold as Bio-Rad MCA1701, MCA1701B; BioLegend 645701, 645702, 645705, 645706; OriGene CF804804, TA804804, TA804804S, TA804804AM, TA804804BM; Santa Cruz Biotechnology sc-126, sc-126 AC, sc-126 X, sc-126 HRP; Abcam ab1101, ab204452, ab237976; Active Motif 39553; Invitrogen MA5-12571, AHO0152; MyBioSource MBS212262; Cell Signaling Technology 18032; MilliporeSigma P6874; BD Biosciences 554293; LifeSpan Biosciences LS-C11553.