Thirty μg of protein from the extracts was loaded per lane. The proteins were separated using sodium dodecylsulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% fat-free dry milk in 1 X Tris-buffered saline containing 0.05% Tween-20 (Sigma, Missouri, USA) for 16 h at 4°C. The blots were probed with primary antibodies against Bax (Becton Dickinson, CA, USA) (1 : 1000 dilution), Bcl-2 (Becton Dickinson, CA, USA) (1 : 1000 dilution), and glyceraldehyde-3-phosphate dehydrogenase (Becton Dickinson, CA, USA) (1 : 1000 dilution) for 1 h and then incubated with horseradish peroxidase-labeled secondary antibody. Protein bands were detected and quantified using an image analyzer (Scientific Imaging Systems V.3.6.3., Kodak Company, Stamford, CT, USA) (Figure 1(a) ).
Bcl2 associated x (bax)
Manufactured by BD
Sourced in United States
Bax is a laboratory equipment product designed for sample preparation and analysis. It is a versatile device that can be used in various scientific and medical applications. The core function of Bax is to facilitate efficient and accurate sample handling and processing.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (13 mentions)
Bcl 2, by BD (12 mentions)
Cleaved caspase 3, by Cell Signaling Technology (10 mentions)
Bcl 2, by Santa Cruz Biotechnology (7 mentions)
Bcl xl, by BD (6 mentions)
Mcl 1, by BD (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Bcl 2, by Agilent Technologies (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Cytochrome c, by Santa Cruz Biotechnology (3 mentions)
Cytochrome c, by BD (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Fibronectin, by Santa Cruz Biotechnology (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
41 protocols using bcl2 associated x (bax)
1
Western Blot Analysis of Bax and Bcl-2
2
Western Blot Analysis of Stem Cell and Cancer Markers
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Whole cell lysates (50 μg) were separated by electrophoresis on 12.5% denaturing polyacrylamide gels. The membranes were incubated overnight at 4°C with primary antibodies (0.1 μg/ml) against Oct4, Nanog, β-AR and phospho EGFR were purchased from Abcam Corporation (Abcam, Cambridge, UK), Snail, Twist, Fibronectin, E-cadherin, CD21, CD44, CD133, ALDH-1, phospho AKT, phospho NFκB, α7-nAChR, Bcl-2 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Bax was obtained from Becton, Dickinson and Company (BD, Transduction Laboratories TM), MDR-1 and ABCG2 were purchased from Millipore Corporation (Millipore Corporation, Billerica, MA, USA), caspase-3 and PARP were obtained from Invitrogen Corporation (Invitrogen, Camarillo, CA, USA) in Tris-Tween-Buffer-Saline buffer containing 3% non-fat milk. Subsequently, each membrane was washed and incubated for 1 h at 25°C with a secondary anti-mouse, anti-rabbit, or anti-goat antibody conjugated with horseradish peroxidase (1: 1000; Santa Cruz Biotechnology, Inc.), as previously reported. [25 (link)]
3
Apoptosis Signaling Pathway Assay
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The materials used in this study were obtained from the following: Dulbecco’s modified Eagle medium (DMEM)/F12, Penicillin-Streptomycin (P/S), 0.25% trypsin-EDTA from Gibco BRL (Grand Island, NY, USA); B-27 supplement, FGF, and Dimethyl sulfoxide (DMSO) from Invitrogen (Carlsbad, CA, USA); EGF from Sigma-Aldrich Co (St. Louis, MO, USA); Tween® 20 and ECLTM Western blotting detection reagent from Amersham Life Science (Arlington Heights, IL, USA); benzalkonium chloride (≥95%), diazolidinyl urea ≥95%), and imidazolidinyl urea (<=100%) from Sigma (St. Louis, MO, USA); BAX from BD Biosciences (BD Biosciences, USA); Bcl-2 from Santa Cruz (CA, USA); anti-β-actin from Sigma; cleaved caspase-3 from Cell Signaling (Boston, MA, USA).
4
Immunohistochemical Analysis of Apoptosis Markers
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Tumor tissue was fixed in 10% formalin, dehydrated, paraffin-embedded, and sectioned into 4-μm blocks. For each animal, a single microscope slide with three sections of the paraffin block was made. The sections were washed three times with PBS for 5 minutes each, blot dried, and then treated with 3% hydrogen peroxide for 30 minutes at room temperature to block endogenous peroxidase activity. The sections were immersed in an antigen-retrieval solution (citrate buffer, pH 6.0) for 10 minutes followed by rinse with PBS. After blocking with normal goat serum for 30 minutes at 37°C, sections were incubated overnight with primary antibodies against BAX, BCL-2, and P53 (BD Biosciences, San Diego, CA) (1:100 dilution in PBS). Following this, the sections were also incubated with a peroxidase-conjugated anti-rabbit IgG secondary antibody (Biosynthesis Biotechnology Co., Ltd.) for 1 hour at room temperature. Subsequently, the sections were incubated with 3,3-diaminobenzidine reagent (ZSGB-BIO, Beijing, P.R. China) for 10 minutes, counterstained with hematoxylin, dehydrated, and mounted for microscopy. The slides were viewed at 100× or 400× magnification with positive cells identified by the brown-stained appearance of the cells. Expression levels were quantified based on the AOD of the positive cells in five fields/sample using the Image-Pro Plus 6.0 software.
5
Quantitative Western Blotting of Apoptosis Regulators
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Total protein extracts were prepared by lysing cells in lysis buffer (20 mM Tris-pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% (v/v) glycerol and 1% (v/v) Triton-X-100; Sigma-Aldrich) with protease inhibitor (Complete Mini, EDTA-free) for 1 h at 4 °C. Lysate supernatants equivalent to 200,000 cells were separated by SDS-PAGE (NuPAGE 4–12% Bis Tris gels, Invitrogen) before transferring to nitrocellulose and probing with antibodies to: MCL-1 (Walter and Eliza Hall Institute, clone 19C4-15), BCL-XL (BD Transduction Laboratory), BCL-2 (BD Biosciences), BFL-1 (Cell Signaling Technology), BAK (Sigma), BAX (BD Pharmingen), BIM (Walter and Eliza Hall Institute, clone 3C5), NOXA (Abcam) and β-actin (Sigma).
6
Triclosan Cytotoxicity Signaling Pathway
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Triclosan was obtained from Sigma (St. Louis, MO, USA) while Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), B-27 supplement and FGF were purchased from Invitrogen (Carlsbad, CA, USA). EGF was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Penicillin/streptomycin, 0.25% trypsin-EDTA, Tween® 20, and ECLTM Western blotting detection reagents were obtained from Amersham Life Science (Arlington Heights, IL, USA).
Antibodies were purchased from the following companies: anti-β-actin from Sigma (St. Louis, MO, USA); caspase3, cleaved caspase 3, ERK, phosphor-ERK, JNK, phosphor-JNK, PI3K, phosphor-PI3K, p38, phosphor-p38, Akt and phosphor-Akt from cell signaling (Boston, MA, USA); Bax from BD PharmingenTM (BD biosciences, USA); Bcl-2 and cytochrome C from Santa Cruz (CA, USA).
Antibodies were purchased from the following companies: anti-β-actin from Sigma (St. Louis, MO, USA); caspase3, cleaved caspase 3, ERK, phosphor-ERK, JNK, phosphor-JNK, PI3K, phosphor-PI3K, p38, phosphor-p38, Akt and phosphor-Akt from cell signaling (Boston, MA, USA); Bax from BD PharmingenTM (BD biosciences, USA); Bcl-2 and cytochrome C from Santa Cruz (CA, USA).
7
Western Blotting Analysis of Apoptosis Markers
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Western blotting analysis was performed as previously described [73 (link)]. Sources of antibodies are: BAX, BNIP3, Mcl-1 and acid ceramidase; Bcl-xL, Bcl-2, Beclin-1 and Cytochrome C: BD Biosciences (San Diego, CA); BAK, BOK, BIM, BAD, PUMA, xIAP and Cathepsin D: Cell Signaling Tech (Danvers, MA); Cathepsin B: Abcam (Cambridge, MA); β-actin: Sigma-Aldrich (St Luis, MO).
8
Antibody Procurement for Cell Signaling
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Antibodies were purchased from different companies: Bim, Bax, Tim23, PKR and the full length of caspase 3 from BD Bioscience (San Jose, CA); β-actin, Tubulin, Bcl-2, Bad and Bak were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DAP3 and 5 antibodies were purchased from Cell Signaling (Danvers, MA). Human RNase L monoclonal antibody was a gift from Dr. Robert Silverman (Cleveland Clinic, OH).
9
Western Blot Analysis of Cellular Proteins
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Cells were harvested and lysed using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, China). Protein concentration was quantified using a Bicinchoninic Acid Protein Assay kit (Sangon Biotech Co., Ltd., Shanghai, China). Total of 10 μg protein were subjected to 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies fibronectin (BD Biosciences), Bax, Bcl-2, DKK3, β-catenin, c-myc, cyclin D1 and β-actin (Cell Signaling Technology, Beverly, MA, USA), followed by further incubation with HRP-labeled secondary antibody (Abcam, Cambridge, MA, USA) for 2 h. The protein signals were visualized using an enhanced chemiluminescence system (ECL™; Amersham, Little Chalfont, UK).
10
Analyzing Fusion Protein Expression
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To determine the expression level of the fusion proteins, SDS-PAGE and Western Blot analysis of cytoplasmic cell lysates was performed. Cell extracts were prepared in lysis buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 10% (v/v) glycerol, 1 mM Na3VO4, 10 mM Na4P2O7, 10 mM NaF, 1 mM EDTA, 10 µg/mL leupeptin, 1 mM PMSF, and 1% (v/v) Triton X-100) and protein content was determined by BCA assay (Thermo Scientific, Carlsbad, CA, USA). After SDS-PAGE electrophoresis, proteins were transferred to nitrocellulose membranes. Proteins were immunodetected by using appropriate primary antibodies and peroxidase-labeled secondary antibodies (Sigma–Aldrich, St Louis, MO, USA). Bands were visualized with Pierce ECL or ECL Plus Western Blotting Substrate (Thermo Scientific). Specific antibodies against the following proteins were used as follows: Bcl-XL (Cell Signaling Technology, Danvers, MA, USA); Bcl-2 (Abcam, Cambridge, UK); Mcl-1 (Santa Cruz Biotechnology, Dallas, TX, USA); Bim (Merck Millipore, Darmstadt, Germany); Puma (Abcam); Noxa (Santa Cruz Biotechnology); Bax (BD Biosciences); and Bak (Santa Cruz Biotechnology and Millipore, Darmstadt, Germany). The protein-loading control was achieved by membrane reprobing with an anti-β-actin or anti-α-tubulin antibody (Sigma–Aldrich).
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