Immunoblotting was carried out as described previously5 (link). Proteins were separated on SDS-PAGE gels, electro-transferred onto polyvinylidene difluoride membranes and incubated overnight at 4 °C with the following antibodies: anti-Atg3, anti-Atg5 (D1G9), anti-Atg7 (D12B11), anti-Beclin (D40C5), anti-caspase-3 (Cell Signaling), anti-Bcl-2 (Millipore), anti-Bax (Millipore), anti-LC3 (Sigma-Aldrich), anti-LXRα (SantaCruz), anti-LXRβ (SantaCruz), anti-Nur77 (Active motif), anti-NOR1 (R&D systems), anti-PARP (Cell Signaling), anti-Vps34 (Cell Signaling), or anti-Actin (Millipore). For visualization, an ECL plus kit (Amersham Biosciences) was used, and chemiluminescence was measured by autoradiography (Supplementary Fig. 9). Specific bands were quantified with ImageQuant software.
Anti bax
Manufactured by Merck Group
Sourced in United States
Anti-Bax is a laboratory reagent used in biochemical research. It is a protein that binds to and inhibits the pro-apoptotic protein Bax, which plays a role in programmed cell death. Anti-Bax is used to study the regulation of apoptosis in various cell types and disease models.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Merck Group (17 mentions)
Anti β actin, by Merck Group (8 mentions)
Anti caspase 3, by Merck Group (7 mentions)
Anti cleaved caspase 3, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anti gapdh, by Merck Group (4 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti parp, by Cell Signaling Technology (3 mentions)
Anti p53, by Merck Group (3 mentions)
Bcl 2, by Merck Group (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (3 mentions)
Anti actin, by Merck Group (2 mentions)
Anti p70s6k, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti tnf α, by Merck Group (2 mentions)
35 protocols using anti bax
1
Immunoblotting Analysis of Autophagy Markers
2
Evaluating Mcl-1 Interactions with Bim and Bak
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Interactions between Mcl-1 and Bim or Bak were evaluated by co-IP analysis. CHAPS buffer (150 mmol/L NaCl, 10 mmol/L HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid] pH 7.4, protease inhibitors, and 1% CHAPS) was employed to avoid artifactual associations [43] (link). Cells were lysed in CHAPS buffer and 200 µg of protein per condition were immunoprecipitated with 1 µg anti-Mcl-1 (Santa Cruz Biotechnology or BD Biosciences), anti-Bak, or anti-Bim (Santa Cruz Biotechnology), followed by Dynabeads (Dynal, Oslo, Norway). IP samples were then subjected to Western blot analysis using anti-Bim (Millipore), anti-Mcl-1, or anti-Bak (Santa Cruz Biotechnology) as primary antibodies, respectively.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.
3
Investigating Oxidative Stress Signaling
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The 4-hydroxynonenal was obtained from Biomol (Plymouth Meeting, PA, USA). Dulbecco's Modified Essential Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). Anti-pAKT, anti-AKT, anti-cleaved-caspase-3, anti-p70S6K, and anti-p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bcl2, anti-GAPDH, and anti-Bax antibodies were from Millipore (Billerica, MA, USA). Other reagents were of the highest purity available.
4
Apoptosis Induction and Mitochondrial Dysfunction
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The following reagents and antibodies were used: Bongkrekic acid (Santa Cruz Biotechnology, La Jolla, CA), JC-1 (eBioscience, San Diego, CA), Cyclosporine A, 2′,7′-dichlorofluorescein acetate (DCFH-DA), N-acetyl cysteine (Sigma, St.Louis, MO), Dioleylphosphatidylserine (Avanti Lipids, Alabaster, AL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ((Roche Diagnostics, Indianapolis, IN), and disuccinyl suberate (Thermo Scientific Fischer, Rockford, IL). Anti-Bcl-2, anti-β-Actin (Abcam, Cambridge, MA), anti-Cyto c (eBioscience, San Diego, CA), anti-AIF, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology, Boston, MA, anti-Survivin, Smac/Diablo, α-Tubulin (Novus biological, Littleton, CO), anti-COX-4, anti-Bax (N-20; Santa Cruz Biotechnology, La Jolla, CA), anti-Bax (polymer-recognizing A67 clone; Sigma, St.Louis, MO) and anti- cleaved PARP (Millipore, Bedford, MA).
Animal maintenance and experimental procedures were carried out in accordance with the US National Institute of health Guidelines for Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and Cincinnati Children's Hospital Medical Center.
Animal maintenance and experimental procedures were carried out in accordance with the US National Institute of health Guidelines for Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and Cincinnati Children's Hospital Medical Center.
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted from liver tissue or cells using lysis buffer [50 mM Tris (pH 8.0), 1% NP-40, 150 mM NaCl, 0.1% SDS]. Quantitative analysis of the protein was performed using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Equal protein quantities (40 µg per lane) were separated by 12% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore). Subsequently, the membranes were blocked with 5% skimmed milk for 1 h at room temperature and probed with anti-Bax (cat no. SAB4502546; 1:1,000, Sigma Aldrich; Merck KGaA), anti-caspase-3 (cat no. 14220; 1:1,000; Cell Signaling Technology, Inc.) or anti-GAPDH (cat no. G9545; 1:2,000; Sigma Aldrich; Merck KGaA) antibodies overnight at 4°C. After 4 washes in PBST (0.1% Tween-20), the membranes were incubated with Horseradish Peroxidase-conjugated Goat Anti-Rabbit IgG H&L pre-adsorbed (1:2,000; cat. no. ab7090; Abcam) for 2 h at room temperature. The immunoreactive proteins were detected using ECL reagent (Pierce; Thermo Fisher Scientific, Inc.).
6
Analysing Cell Protein Expression
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H1299, HCT116 (p53+/+), HCT116 (p53−/−) and Hwtp53 (expressing wtp53, induced with 1 μg/ml tetracycline for 20 h) [44 (link)] cells were harvested from 10 cm plates and lysed with 1× PLB (Promega), followed by the sonication of cells (Bandelin Sonopuls). Samples (100 μg of total protein) were analysed on 12.5% SDS-PAGE gels and proteins were detected by the following primary antibodies: DO1 (anti-p53, kindly provided by B. Vojtesek), anti-cyclin-dependent kinase inhibitor 1A (CDKN1A; Millipore), anti-β-Actin (Sigma), anti-BAX (Sigma), anti-MYC (Cell Signaling).
7
Melatonin-Based Anti-Apoptotic Assay
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Melatonin (N-acetyl-5-methoxytryptamine), soybean oil, Bradford reagent, PMSF, BSA, propidium iodide, protease inhibitor cocktail, anti-p53, anti-Bax, anti-Bcl-x,
HRP-conjugate and formalin were procured from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. EGTA, tris-HCL, trichloroacetic acid, tween-20, tween-100, skimmed milk powder, and phosphate buffer saline (PBS) were purchased from HiMedia, Mumbai, India. EDTA, NaCl ABTS
(2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate), and ethanol were from Merck, Germany. ECL chemiluminescence reagent was from Amersham Pharmacia Biotech, Piscataway, NJ, USA. Dimethyl sulfoxide
(DMSO) and sodium dodecyle sulphate were from Calbiochem, San Diego CA USA. DNase free RNase was procured from Genei, Bangalore, India.
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Melatonin (N-acetyl-5-methoxytryptamine), soybean oil, Bradford reagent, PMSF, BSA, propidium iodide, protease inhibitor cocktail, anti-p53, anti-Bax, anti-Bcl-x,
HRP-conjugate and formalin were procured from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. EGTA, tris-HCL, trichloroacetic acid, tween-20, tween-100, skimmed milk powder, and phosphate buffer saline (PBS) were purchased from HiMedia, Mumbai, India. EDTA, NaCl ABTS
(2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate), and ethanol were from Merck, Germany. ECL chemiluminescence reagent was from Amersham Pharmacia Biotech, Piscataway, NJ, USA. Dimethyl sulfoxide
(DMSO) and sodium dodecyle sulphate were from Calbiochem, San Diego CA USA. DNase free RNase was procured from Genei, Bangalore, India.
8
Cytotoxicity Evaluation of Cell Lines
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The cell culture medium (RPMI 1640), fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Gibco BRL (life technolologies, Paisley, Scotland). The culture plates were obtained from Nunc (Denmark). Hoechst 33285 was purchased from Sigma Chemical Company (Germany). MTT assay kit was purchased from Roche (Germany). All antibodies, except β actin, including anti-Bcl2 and anti-Bax were purchased from Sigma (St Louis, MO, USA). β actin antibody was purchased from Alexis Biochemicals (San Diego, CA). Annexin-V-FITC kit was purchased from IQ product (Groningen, The Netherlands). All cell lines were obtained from Pasteur Institute of Iran (Tehran).
9
Cell Viability and Apoptosis Assay
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10
Protein Expression Analysis in Neurons and Astrocytes
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Total protein extracted from primary neurons or astrocytes was used for western blotting. The membranes were incubated in primary antibodies including anti-IL6R (1:400, Abcam, USA), anti-STAT3 (1:800, ABZOOM, USA), anti-Bax (1:200, Sigma, USA), anti-Bcl-2 (1:800, Sigma, USA) and anti-β-actin (1:500, Santa Cruz, USA) at 4 °C overnight, followed by incubation in HRP-conjugated secondary antibodies (Santa Cruz, USA) at room temperature for 1 h. Then, the protein bands were detected using a chemiluminescent HRP substrate (Millipore, USA). The images were captured using a Syngene GBox Imaging System.
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