After ultrasound treatments, cells were lysed in the lysis buffer by extracting proteins. After centrifugation, supernatant was taken as total protein. Bradford method was used for protein quantification. The same amount of protein extract was isolated by 10% SDS-PAGE and transferred to PVDF membranes (0.45μm, Millipore). The blots were blocked with 5% milk, then incubated overnight at 4° C with primary antibodies against p-γ-H2AX (Cell Signaling Technology, USA), cleaved-caspase3 (Cell Signaling Technology), Bax and Bcl-2 (Cell Signaling Technology), XPD (Cell Signaling Technology), PARP-1(Cell Signaling Technology), pADPr (Abcam, USA), β-actin (Cell Signaling Technology), β-tubulin (Cell Signaling Technology). Thereafter, these blots were incubated with HRP-conjugated secondary antibody, developed in ECL solution, and exposed onto hyper film (Amersham Biosciences, UK) for 1-10 min. The Image J software (NIH) was used to analyze the gray value of all bands.
Bax and bcl 2
Manufactured by Cell Signaling Technology
Sourced in United States
Bax and Bcl-2 are proteins involved in the regulation of apoptosis, or programmed cell death. Bax is a pro-apoptotic protein that promotes cell death, while Bcl-2 is an anti-apoptotic protein that inhibits cell death. These proteins are often used in research to study the mechanisms of cell death and survival.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin, by Bio-Rad (2 mentions)
Hyperfilm, by Cytiva (1 mentions)
P γh2ax, by Cell Signaling Technology (1 mentions)
Padpr, by Abcam (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Caspase 9, by Proteintech (1 mentions)
Akt and phosphorylated akt, by Abcam (1 mentions)
Mtor and phosphorylated mtor, by Bioworld Technology (1 mentions)
Human β actin, by Transgene (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ab6789, by Abcam (1 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
Ab6721, by Abcam (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
5 protocols using bax and bcl 2
1
Evaluating Cellular Responses to Ultrasound Treatment
2
Western Blot Analysis of Transfected Cells
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Transfected cells were harvested 48 h after transfection. Cells were lysed in RIPA buffer (Heart, Beijing, China). Total protein samples were separated by SDS‐PAGE polyacrylamide gel and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were immunological overnight at 4°C with primary antibodies as follows: PTEN (1:500; WanleiBio, Shenyang, Liaoning, China); Akt and phosphorylated‐Akt (phosphoresce on S473; 1:500, Abcam, San Francisco, CA, USA); mTOR and phosphorylated‐mTOR (phosphorylated on S2998; 1:1000, Bioworld, Nanjing, Jiangsu, China); Bax and Bcl‐2 (1:1000, Cell Signaling, Danvers, MA, USA); caspase‐9 (1:500, Protein Tech, Wuhan, Hubei, China); and human β‐actin (1:5000; Transgene, China). PVDF membranes were washed with TBST and then incubated with a secondary antibody, HRP‐conjugated goat IgG (1:5000; Transgene, China) for 1 h at 37°C. Signals were detected by the Bio‐Rad Gel imaging system. The images were quantified by Quantity One (Bio‐Rad, USA), and relative protein expression was normalized to β‐actin levels in each sample. All experiments were performed in triplicate.
3
Quantitative Protein Analysis of Cell Signaling
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The detailed protein extraction was described elsewhere (Yang et al., 2019 (link)). In brief, the total protein level was quantified using a protein assay kit (Bio-Rad, USA). Equivalent amount of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by being transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with primary antibodies, including NF-κB and Phospho-NF-κB, SIRT1, Bax and Bcl-2, cleaved caspase-3 (1:1,000, Cell Signaling Technology, Danvers, MA, USA), inducible nitric oxide synthase (iNOS), IL-10 (1:1,000, Abcam, Cambridge, UK), and GAPDH (1:1,000, T0004-HRP, Affinity), at 4°C overnight, followed by HRP-conjugated secondary antibodies (1:5,000, ab6721 or ab6789, Abcam, USA) for 1 h at 25°C. Quantification of the protein bands was carried out by using the Image Analyses Software (ChemiDoc™ MP Imaging System Bio-Rad, USA).
4
Investigating Apoptotic and Necroptotic Signaling
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MDA-MB-231 and MCF-7 cells were lysed in RIPA lysis buffer on ice for 30 min, then centrifuged (12000 g/min; 30 min) at 4°C. A bicinchoninic acid (BCA) assay was used to detected protein concentrations. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (millipore, USA), and then incubated with primary antibodies overnight at 4°C after the membranes were blocked (5% skim milk in PBS with 0.1% Tween 20) for 4 h. The next day, the membranes were imaged with gel imaging equipment (Bio-Rad, USA) after the membranes were incubated with secondary antibodies for 2 h. β-actin was used as a loading control. The following antibodies were used: Bcl-2 and Bax (Cell Signaling technology, USA); anti-RIP1, anti-RIP3, and p-RIP3 (Santa Cruz Biotechnology, USA); TNF-α (Abcam, USA); Caspase 3 (Enzo, USA); Ppm1b (BETHYL, USA); anti-β-actin (Biosharp, China) All reagents were dissolved according to the manufacturer’s instructions.
5
Western Blot Analysis of Apoptotic Proteins
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Cells were homogenized in ice-cold RIPA Tissue Protein Extraction Reagent (Beyotime, China) supplemented with 1% proteinase inhibitor mix. The proteins were separated by SDS-PAGE and then electrotransferred to Hybond-P PVDF membrane (Millipore, France). The membrane was incubated with appropriate primary antibodies overnight at 4°C. Antibodies used were Bcl-2 and Bax (1:500, Cell Signaling Technology, USA), Cyt C and β-actin (1:500, Beyotime, China), VDAC (1:1,000, Cell Signaling Technology, USA). Immunoreactivity was visualized by second horseradish peroxidase-conjugated antibody (1:5,000, Sigma, USA) and enhanced chemoluminescence (Beyotime, China). Quantified densitometric analysis was using with UVP BioSpectrum Multispectral Imaging System (Ultra-Violet Products Ltd. USA).
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