Protein lysates were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40 (Beyotime, ST366) with protease inhibitor cocktail (Cell Signaling Technology, 5871) and were used for an overnight immunoprecipitation (500 μg per sample) at 4°C with 10 μL of the following antibodies: anti-FLAG (GenScript, L00425); anti-TP53 (Santa Cruz Biotechnology, sc-47698); anti-HMGB1 (Santa Cruz Biotechnology, sc-26351) and anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402). A total of 20 μL of protein A/G Plus-agarose (Santa Cruz Biotechnology, sc-2003) was included in the incubation with the anti-TP53, anti-HMGB1 and anti-SQSTM1 antibodies. The resulting immunoprecipitates were analyzed using western blot assays.
Anti tp53
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-TP53 is a laboratory reagent used for the detection and quantification of the TP53 protein in biological samples. TP53, also known as p53, is a tumor suppressor protein that plays a crucial role in regulating cell growth and division. The Anti-TP53 reagent can be used in various research and diagnostic applications, such as Western blotting, immunohistochemistry, and ELISA, to analyze the expression and function of TP53 in different cell and tissue types.
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Lab products found in correlation
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti sqstm1, by Santa Cruz Biotechnology (3 mentions)
Anti hmgb1, by Santa Cruz Biotechnology (3 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Anti flag, by GenScript (1 mentions)
Np 40, by Beyotime (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti acetylated histone h3, by Merck Group (1 mentions)
Sc 6243, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3, by Merck Group (1 mentions)
Anti h3k27ac, by Active Motif (1 mentions)
Anti h3k4me2, by Cell Signaling Technology (1 mentions)
Anti lsd1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti atf4, by Cell Signaling Technology (1 mentions)
10 protocols using anti tp53
1
Immunoprecipitation and Western Blot Analysis
2
Protein Expression Analysis by Western Blot
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Whole-cell protein lysates were prepared using Mammalian Protein Extraction Reagent (Thermo Scientific, Oslo, Norway) supplemented with phosphatase and protease inhibitors. To ensure complete disruption of cells, sonication was performed. After 2–3 h of incubation at 4 °C, the lysates were centrifuged at 15 000 r.p.m. for 15 min. The supernatant was aspirated and protein concentration was measured using a bicinchoninic acid assay (Thermo Scientific). Protein expression levels were evaluated by western blots, applying 20 μg protein onto 4–20% SDS gels and overnight incubation at 4 °C with the antibodies anti-acetylated histone H3 (polyclonal; Merck Millipore, Billerica, MA, USA), anti-total histone H3 (clone A3S; Merck Millipore), anti-HIF1 A (clone 54; BD), anti-EPAS1 (HIF2α) (polyclonal; Novus Biologicals, Littleton, CO, USA) and anti-TP53 (polyclonal, sc-6243; Santa Cruz, Dallas, TX, USA). The mouse monoclonal anti-γ-tubulin (anti-TUBG1) (clone GTU-88; Sigma-Aldrich) or anti-MCM7 (clone DCS-141, Santa Cruz) was used as loading control. Secondary antibodies were peroxidase-conjugated goat anti-rabbit and donkey anti-mouse (both from Jackson ImmunoResearch Laboratories, West Grove, MA, USA). For visualisation, chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) or LumiGLO Chemiluminescent Substrate (KPL, Gaithersburg, MD, USA) was used.
3
Western Blot Antibody Validation
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Anti-LSD1 (Cell Signaling Technology, catalog 2139), anti-TP53 (Santa Cruz Biotechnology, catalog sc-126), anti-p21(CDKN1A) (Cell Signaling Technology, catalog 2947), anti-GAPDH (Santa Cruz Biotechnology, catalog sc-32233), anti-H3K4me2 (Cell Signaling Technology, catalog 9725), anti-H3K27Ac (Active Motif, catalog 39133), and anti–Histone H3 (MilliporeSigma, catalog 06-755) were used for protein detection by Western blotting.
4
Immunoblot Analysis of Apoptosis Regulators
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Antibodies used were: anti-ATF3 (Santa Cruz, #sc-188), anti-ATF4 (Cell Signaling, #11815), anti-BAX (Cell Signaling, #2772), anti-BAK (Cell Signaling, #3814), anti-BCL-2 (Cell Signaling, #15071), anti-MCL-1 (Cell Signaling, #5453), anti-NOXA (Merck, #OP180), anti-TP53 (Santa Cruz, #sc-126), anti-eIF2α (Cell Signaling, #9722), anti-(P)-eIF2α (Cell Signaling, #9721), anti-GAPDH (Cell Signaling, #2118), anti-β-ACTIN (Merck, #A5541). Secondary anti-mouse (#7076 S) and anti-rabbit (#7074 S) horseradish peroxidase-coupled antibodies were from Cell Signaling.
5
Comprehensive Western Blot Analysis
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Western blot analyses were performed as previously described with lysates from fresh tissues and cells [62 (link)]. The primary antibodies used were anti-EZH2 (1:1000, Cell Signaling Technology), anti-GAPDH (1:1000, Santa Cruz Biotechnology), anti-β-catenin (1:1000, Santa Cruz Biotechnology), anti-c-myc (1:500, Santa Cruz Biotechnology), anti-cyclin D1 (1:500, Santa Cruz Biotechnology), anti-GSK-3β (1:1000, Santa Cruz Biotechnology), anti-TP53 (1:500, Santa Cruz Biotechnology), anti-H3K27me3 (1:1000, Cell Signaling Technology), and anti-histone 3 (1:1000, Cell Signaling Technology). The secondary antibodies used included horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Thermo Fisher Scientific, New York, NY, USA).
6
Immunohistochemistry and Elastic Staining of FFPE Tissues
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FFPE tissues were sectioned at 5 mm thickness and analyzed by IHC and EVG staining. The following antibodies were used: anti-TP53 (Santa Cruz Biotechnology, Dallas, TX, USA, catalog number: sc-47698), anti-CDKN2A/p16 (Roche Diagnostics, Cat #6695221001), anti-SMAD4 (Santa Cruz Biotechnology, Cat #sc-7966), anti-CD4 (Leica Biosystems, Wetzlar, Germany, Cat #CD4-368-L-CE), anti-CD8 (Roche Diagnostics, Cat #5493846001), anti-FOXP3 (Abcam, Cambridge, UK, Cat #ab20034), anti-CD45RO (BioGenex Laboratories, San Ramon, CA, USA,Cat #AM113-5M), anti-CD68 (ProteinTech Illinois, USA, Cat #66231-2-Ig), anti-CD206 (ProteinTech Illinois, USA, Cat #60143-1-Ig) and anti-α-SMA (Santa Cruz Biotechnology, catalog number: sc-53142). EVG staining was performed using an Elastic Stain Kit (Abcam, Cat #ab150667) according to the manufacturer’s protocol.
7
Western Blot Protein Analysis Protocol
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Whole cell extraction was conducted using PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Equal amounts (20 μg) of each sample were separated on 8-15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 hr at room temperature, washed with TBST, and subsequently incubated with primary antibodies: anti-S100A14 (Proteintech, Chicago, IL), anti-phospho-Akt (ser473), anti-Akt, anti-phospho-Erk1/2, anti-Erk1/2 (Cell Signaling, Danvers, MA), anti-TP53 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH (Santa Cruz Biotechnology). Primary antibodies against each protein were detected by secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, Munich, Germany). Specific bands for each protein were detected on AGFA X-ray film (Agfa Health Care, Mortsel, Belgium) using the SuperSignal Chemiluminescence kit (Thermo Scientific, Rockford, IL).
8
Protein Expression Analysis of HosDXR150 Cells
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Treated and untreated HosDXR150 cells were lysed in 50 mM Tris/HCl, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton-X, 0.1 mM Na3VO4 plus protease inhibitors (Roche Molecular Biochemicals, Germany). Equal amounts of proteins were resolved on a 7% or 10% SDS-PAGE, transferred onto nitrocellulose membranes, and then successively blocked with 5% non-fat dry milk. Anti-RBL2 was from BD Transduction Laboratories. Anti-FAS, anti-CASP10 and anti-IL12A were from Sigma-Aldrich. Anti-TP73, anti-TP53, anti-actin and the horseradish peroxidase secondary antibodies were from Santa Cruz Biotechnology. Primary antibodies were used at a dilution of 1∶200. Secondary antibodies were used at a dilution of 1∶5000. Signals were acquired using the ImageQuant LAS 4000 (GE Healthcare).
9
Western Blot Analysis of Cellular Proteins
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Protein lysates (30–50 μg per sample) were separated on 8, 10 or 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Merck Millipore, IPVH00010). Incubations with primary and secondary antibodies and signal detection followed the manufacturer’s protocol using Western Lightning Plus-ECL (PerkinElmer, NEL105001EA). Quantitative image analysis was performed by using Quantity One software. The following antibodies were used: anti-GAPDH (Santa Cruz Biotechnology, sc-25778), anti-TP53 (Santa Cruz Biotechnology, sc-47698, sc-6243), anti-HMGB1 (Santa Cruz Biotechnology, sc-26351), anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402), anti-LC3B (Cell Signaling Technology, 2775s), anti-SQSTM1 (Cell Signaling Technology, 5114s), anti-HMGB1 (Cell Signaling Technology, 6893s), anti-ATG7 (Cell Signaling Technology, 2631s), anti-ATG5 (Cell Signaling Technology, 8540s), anti-cleaved-PARP (Cell Signaling Technology, 5625s), and HRP-labeled secondary antibodies (MultiSciences Biotech, GAR007, GAM007, and RAG007). The signal intensity was quantified using Quantity One® software and was shown to be within the linear range of detection.
10
Immunofluorescence Staining of Cellular Proteins
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Cells grown on poly-D-Lysine-coated cover slips were washed with phosphate-buffered saline (PBS; Gibco, 10010023) and fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS for 15 min at room temperature. They were then permeabilized with 0.2% Triton X-100 (BioFROXX, 1139ML100) in PBS for 15 min, blocked with nonfat dried milk in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) with Tween-20 (Aladdin, T104863) (TTBS) for 1 h and incubated with primary antibodies at room temperature for 2 h or 4°C overnight. After washing with PBS, the cells were incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies (Thermo Fisher Scientific, A21202, A10042, A10037, A11057) for 2 h, and then stained with DAPI (Dojindo, D212) for 5 min and mounted for fluorescence microscopy. The following primary antibodies were used: anti-TP53 (Santa Cruz Biotechnology, sc-47698), anti-HMGB1 (Santa Cruz Biotechnology, sc-26351), anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402) and anti-LC3B (Cell Signaling Technology, 2775s).
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