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.
Gel imaging equipment
Manufactured by Bio-Rad
Sourced in United States
Gel imaging equipment is a device used to capture and analyze images of electrophoresis gels. It typically includes a camera, light source, and software for image processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Caspase 3, by Enzo Life Sciences (1 mentions)
Tnf α, by Abcam (1 mentions)
Bax and bcl 2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Biosharp (1 mentions)
β actin, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Uhrf1, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Gapdh, by Proteintech (1 mentions)
Quantione imaging software, by Bio-Rad (1 mentions)
Anti rabbit secondary antibody, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca assay, by Beyotime (1 mentions)
Six well culture dishes, by Corning (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Protein extraction kit, by Boster Bio (1 mentions)
8 protocols using gel imaging equipment
1
Investigating Apoptotic and Necroptotic Signaling
2
Western Blot Analysis of Epithelial-Mesenchymal Transition
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FaDu cells and tissue were collected and resuspended in cell lysis buffer for western blot on ice. BCA protein quantification kit was used to detect the protein concentration. Protein from each sample was loaded onto an SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA). Membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.1% Tween-20 (PBS-T). To detect the target proteins, membranes were incubated with primary antibodies Vimentin (1:2000, Proteintech Group), E-cadherin (1:5000, Proteintech Group), GAPDH (1:3000, Proteintech Group), ZEB1 (1:500, Proteintech Group), ARHGDIB (1:5,000; Abcam), UHRF1 (1:500; Abcam), CCN1 (1:1,000; Abcam) and β-actin (1:2,000; Abcam) overnight at 4°C. Membranes were then washed for 10 min with PBS-T for three times and incubated with secondary antibody (1:2,000; Abcam) for 2 h at room temperature. The membranes were washed and developed using chemiluminescence reagents (Millipore) and visualized with gel imaging equipment (Bio-Rad, USA).
3
Western Blot Analysis of Cellular Proteins
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Total proteins were isolated from the cells with protein lysis buffer. The concentrations of the protein samples were determined by bicinchoninic acid (BCA; Thermo Scientific) protein assay. Proteins samples (40 μg) were run and separated on a 10% of SDS-polyacrylamide gel (SDS-PAGE), and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking in blocking buffer (1X TBST with 5% w/v de-fatted milk powder), the membranes were incubated with specific primary antibodies at 4°C overnight. Primary antibodies against Skp2 (1:1,000), ZO-1 (1:1,000), N-cadherin (1:1,000), E-cadherin (1:2,000), Slug (1:1,500), Vimentin (1:1,000), Nanog (1:1,000), Oct4 (1:1,500), ABCB1 (1:1,500), Foxo1 (1:1,500) and p21 (1:1,000) were used. The membranes were then washed with TBST and probed with anti-mouse (Cat. no. #A3682, 1:4,000, Sigma-Aldrich, St. Louis, MO, USA) or anti-rabbit secondary antibodies (cat. no. A16110 1:3,000, Thermo Fisher Scientific) at room temperature for 1 h. Finally, the membranes were washed again and detected using enhanced chemiluminescence substrate (ECL) (Sigma-Aldrich; EMD Millipore). Quantitative analysis was carried out using QuantiOne imaging software with gel imaging equipment (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Protein Expression Analysis Protocol
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The cells were plated in six-well culture dishes (Corning Life Sciences) at a density of 4 × 105 cells/ml/well. Following 24 h of incubation, the cells were washed with 1 Ml PBS/well and were harvested using trypsin. Harvested cells were centrifuged and resuspended in lysis buffer. Following incubation on ice for 30 min, the homogenate was centrifuged at 12 000 r.p.m. for 30 min at 4 °C. Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Beyotime Institute of Biotechnology, Beijing, China). Subsequently, 40 μg of protein were separated using 10~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The blotted membranes were blocked with 5% skim milk for 2 h and probed with primary antibodies overnight at 4 °C. The membranes were washed and probed with secondary antibodies for 1 h. The membranes were imaged with gel imaging equipment (Bio-Rad, Hercules, CA, USA). β-actin was used as the loading control.
5
Western Blot Analysis of Ischemic Cortex Proteins
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After reperfusion for 24 hours, ischemic cortex was collected for total protein extraction (n = 3 each group), according to the manufacturer's instructions for the protein extraction kit (Boster). Equivalent amounts of protein were loaded onto 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels for standard electrophoresis. Afterwards, gels were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline for 1 hour at room temperature, and then incubated overnight at 4°C with the following antibodies: monoclonal phospho-JAK2 (Cell Signaling Technology, Danvers, MA, USA; 1:1,000), monoclonal phospho-STAT3 (Cell Signaling Technology; 1:1,000), polyclonal Bax (Boster; 1:500), polyclonal caspase-3 (Boster; 1:500), polyclonal Bcl-2 (Boster; 1:500), and an internal control, monoclonal beta-actin antibody (Cell Signaling Technology; 1:1,000). Next, membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:1,000; Nantong, China) for 1 hour at room temperature. Protein bands were quantified by scanning of visualized bands using enhanced chemiluminescence (Millipore, Bedford, MA, USA) and gel imaging equipment (Bio-Rad, Hercules, CA, USA). Band optical density was measured using Quantity One 4.6.2 software (Bio-Rad).
6
Hispidulin Concentration-Dependent Protein Expression
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CNE-2Z cells were treated with different concentrations of hispidulin (25, 50, and 100 μM) for 48 h. After treatment, cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer for 30 min on ice. Following 12,000 rpm at 4 °C for 15 min, the supernatants were harvested for protein quantification using a bicinchoninic acid assay kit. Then, equal amounts of total protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk for 2 h at room temperature. Then, the PVDF membranes were immunoblotted with specific antibody (Akt, Bax, Bcl-2, HIF-1α, MMP-2, MMP-9, TIMP1, or β-actin) overnight at 4 °C, followed by incubating with the corresponding goat anti-rabbit or anti-mouse antibodies at room temperature for 2 h. Protein levels were measured by an enhanced chemiluminescence kit (Millipore, Burlington, MA, USA) and visualized by gel imaging equipment (Bio-Rad, Hercules, CA, USA).
7
Western Blot Protein Analysis Protocol
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Western blot analysis was carried out as described by Alasmari et al. [41 (link)]. Briefly, isolated proteins (30–50 μg) were electrophoresed on SDS-PAGE gels and then transferred onto PVDF membranes. After blocking with 5% nonfat dry milk for 60 min, blots were probed overnight at 4 °C with selective primary antibodies (against Ho-1, Nrf2, TNF-α, IL6, NF-κB-p65, cleaved-caspase-3, Bcl-2, Bax, and GAPDH, dilution 1:1000). The membranes were subsequently washed, then incubated for 60 min at room temperature with suitable HRP-conjugated secondary antibodies (dilution: 1:5000). The ECL reagent kit and gel imaging equipment (Bio-Rad, Hercules, CA, USA) were used to detect the presence of proteins on the membrane.
8
Protein Analysis by Western Blotting
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Cells were harvested and homogenized in RIPA lysis buffer for 30 min on ice, the lysates were centrifuged at 12,000 × g for 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. After the membranes were blocked with 5 % skim milk in PBS with 0.1 % Tween20 for 4 h, they were incubated overnight at 4 °C with primary antibodies, followed by incubation with the corresponding secondary antibodies. The membranes were imaged with gel imaging equipment (Bio-Rad, USA). β-actin was used as a loading control.
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