Protein samples were prepared in lysis buffer (HEPES 25 mmol/L, KAc 150 mmol/L, EDTA pH 8.0 2 mmol/L, NP-40 0.1%, NaF 10 mmol/L, phenylmethylsulfonyl fluoride (PMSF) 50 mmol/L, aprotinin 1 µg/µl, pepstatin 1 µg/µl, leupeptin 1 µg/µl, and DTT 1 mmol/L). Protein concentration was quantified by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China) according to the protocol using bovine serum albumin (BSA) to prepare a standard curve. Gel electrophoresis was performed with 10–20 µg of protein using 4%–15% gels (Beyotime, China), followed by transblotting to 0.2-µm nitrocellulose membrane (Amersham, Piscataway, NJ, USA). Protein band intensities were determined and detected with BeyoECL Star (Beyotime, China) using the Amersham Imager 680 system (GE). Primary antibodies used in the experiments, including anti-Bcl-2 (rabbit mAb, Cell Signaling, Danvers, MA, USA) and β-actin (rabbit mAb, Cell Signaling, USA) were diluted 1:1,000 in 1% BSA. The secondary antibody used in the experiments including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beyotime, China) was diluted at 1:1,000.
Amersham imager 680 system
Manufactured by GE Healthcare
Sourced in United States, China
The Amersham Imager 680 system is a versatile imaging solution designed for protein and nucleic acid detection and analysis. It utilizes a high-resolution CCD camera and advanced imaging technology to capture and analyze a wide range of fluorescent and chemiluminescent signals. The system is capable of performing a variety of imaging techniques, including western blotting, dot blotting, and gel imaging.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse igg, by Bio-Rad (3 mentions)
Goat anti rabbit igg, by Bio-Rad (3 mentions)
β actin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
0.2 m nitrocellulose membrane, by Cytiva (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
α tubulin, by Merck Group (1 mentions)
Nb100 2324, by Novus Biologicals (1 mentions)
Bace1, by Santa Cruz Biotechnology (1 mentions)
Presenilin 1, by Santa Cruz Biotechnology (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
13 protocols using amersham imager 680 system
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of ER Stress Markers
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Standard Western blot protocol was used as described previously (59 (link)). Protein samples were subjected to SDS-PAGE and transferred to supported nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were stained with ponceau S (Sigma-Aldrich) for protein detection, then blocked with blocking buffer (5% bovine serum albumin in Tris Buffered Saline with Tween 20) and probed with primary antibodies against APP (abcam, Y188), β-actin (Sigma-Aldrich, Clone AC-15), α-tubulin (Sigma-Aldrich, T5168), GM130 (BD Biosciences, Clone 35), PDI (Cell Signaling, C81H6), IRE1 (Novus biological, NB100-2324), phosphor-IRE1 (Novus biological, NB1002323), OASIS (Santa Cruz Biotechnology, sc-514635), presenilin-1 (Santa Cruz Biotechnology, sc-365450), BACE1 (Santa Cruz Biotechnology, sc-33711). Membranes were then probed with horseradish peroxidase–conjugated secondary antibodies (GE Healthcare) and visualized by ECL (SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific). Blots were quantified using the CCD-based Amersham Imager 680 system (GE Healthcare Life Sciences) and the intensity of bands was measured using Image J.
3
Rat Gastric Tissues Protein Analysis
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Rat gastric tissues (50 mg) were homogenized and lysed in ice-cold RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitor and protease inhibitor cocktail. Subsequently, the samples were centrifuged at 10 000 g and 4 °C for 10 min. After centrifugation, the supernatant was collected and subjected to a BCA Protein Assay kit to measure protein concentration. The samples were denatured with reducing SDS sample buffer and boiled in water at 95 °C for 10 min. Subsequently, Western blot analysis was applied to the examination of Keap1, Nrf2, HO-1, NF-κB p65, p-IKKα/β, Bcl2, Bax, Caspase3 and PCNA protein expression. The same amount of protein from all lysates of each sample was separated by 10%−15% SDS-PAGE, and then transferred onto the PVDF membranes, which was incubated overnight with the corresponding primary antibody at 4 °C, followed by the incubation of a secondary antibody. Thereafter, the proteins were measured with Omni-ECL™ Pico light chemiluminescence detection reagents (Epizyme Biomedical Technology Co., Ltd., Shanghai, China) and Amersham Imager 680 system (General Electric, USA). All Western blot studies were repeated three times.
4
Western Blot Protein Detection Protocol
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Methods for western blotting have been described previously.28 Briefly, lysates from whole cells were resolved in SDS‐PAGE. Proteins separated electrophoretically using 12.5% SDS‐PAGE gels were transferred to polyvinylidene difluoride membranes (GE Healthcare) using a wet blotting procedure and incubated with rat anti‐PSF1 (Genestem), or mouse anti‐GAPDH (Millipore). Proteins were detected using horseradish‐peroxidase‐conjugated goat anti‐rat IgG, or goat anti‐mouse IgG (Jackson Laboratories) secondary antibodies and ECL reagents (GE Healthcare). The blots were scanned with an imaging densitometer Amersham Imager 680 system (GE Healthcare).
5
Western Blot Analysis of Cell Lysate Proteins
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The protein concentrations of cell lysate were determined by the BCA protein assay using bovine serum albumin as a standard (Thermo, MA, USA). An equal amount of protein (10 μg) per sample was separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with 4% non-fat milk in TBST buffer at room temperature for 2 h, followed by incubation at 4 °C overnight with primary DRP1 (Cell Signaling Techchnology, MA, USA; dilution 1:2000), IDH1 (Cell Signaling Techchnology; dilution 1:2000), IDH3A (Cell Signaling Techchnology; dilution 1:2000), and β-actin (Sigma; dilution 1:4000) antibodies. After washing with TBST three times, the membranes were incubated with secondary goat-anti-rabbit IgG (Bio-Rad Laboratories, CA, USA; dilution 1:3000) or goat-anti-mouse IgG (Bio-Rad, dilution 1:3000) conjugated with horseradish peroxidase in 4% non-fat milk for 2 h at 37 °C. Membranes were then washed with TBST three times and incubated with enhanced chemiluminescence western blotting substrate (Thermo), followed by visualisation using an Amersham Imager 680 system (GE Healthcare, MA, USA). The level of β-actin was used as an internal control.
6
Western Blot Analysis of Proteins
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Different proteins in whole-cell lysates were separated by 10% SDS‒PAGE and then transferred to PVDF membranes. The PVDF membrane was then cropped to blot different proteins in the same lane. After blocking with 5% (w/v) nonfat dried milk, the membranes with targeted proteins were incubated with the corresponding primary antibodies overnight at 4 °C. The membranes were then washed three times using PBS supplemented with 0.05% Tween-20 (PBST) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.5‰ Tween-20) and incubated with the corresponding horse radish peroxidase-conjugated secondary antibodies at room temperature for 30 min. After washing three times with PBST, the membranes were treated with a chemiluminescent substrate and imaging was performed using an Amersham imager 680 system (GE).
7
Western Blot Protein Detection
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The different proteins of interest in whole cell lysates were separated using 10% SDS-PAGE with pre-stained molecular protein standards; and then transferred to a PVDF membrane. The PVDF membrane was then cropped to blot different proteins in the same lane. After blocking with 5% (w/v) non-fat dried milk, the membranes with targeted proteins were incubated and detected with the corresponding primary antibodies overnight at 4°C. The membranes were then washed three times using PBS plus 0.05% Tween-20 (PBST) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 and 0.5‰ Tween-20) and incubated with HRP-conjugated secondary antibody at room temperature for 30 min. After washing three times with PBST, the membranes were treated with the chemiluminescent substrate, and imaging was performed using the Amersham imager 680 system (GE, CA, USA).
8
HeLa Cell Protein Expression Analysis
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HeLa cells were treated with either 6-methoxyflavone (65 lM) or 0.16% DMSO for 48 h, the cells were lysed for 20 min in cell lysis buffer (Beyotime) containing 1 mM phenylmethanesulfonyl fluoride (Beyotime). The lysate was centrifuged at 14,000 Â g for 5 min at 4 C. Total protein samples were obtained from the supernatants of HeLa cell lysates. Protein concentrations were detected using an enhanced bicinchoninic acid protein assay kit (Beyotime). Subsequently, protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). After blocking with Tris-buffered saline-Tween 20 solutions containing 5% bovine serum albumin for 1 h at 25 C, the membranes were incubated with the primary antibodies overnight at 4 C. The primary antibodies used were: GAPDH, PERK (Sangon Biotech), phospho-PERK (Thr982), EIF2a, phospho-EIF2a (Ser51), ATF4, and CHOP rabbit antibodies (Beyotime). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Sangon Biotech) for 1 h at 25 C. Finally, the protein bands were visually detected with an ultra-sensitive efficient chemiluminescence kit (Beyotime) using the Amersham Imager 680 system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
The functional role of the core gene biomarkers
The functional role of the core gene biomarkers
9
Western Blot Analysis of OPA1 Protein
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The protein concentrations of cell lysate were determined by the BCA protein assay using bovine serum albumin (BSA) as a standard (Thermo, MA, USA). An equal amount of protein (10 µg) per sample was separated on 10% or 15% SDS polyacrylamide gels and then transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 4% non-fat milk in TBST buffer at room temperature for 2 hours, followed by incubation at 4°C overnight with primary OPA1 ( antibodies. After washing with TBST three times, the membranes were incubated with secondary goatanti-rabbit IgG (Bio-Rad Laboratories, CA, USA; dilution 1:3000) or goat anti-mouse IgG (Bio-Rad, dilution 1:3000) conjugated with horseradish peroxidase in 4% non-fat milk for 4 hours at 37°C. Membranes were then washed with TBST three times and incubated with enhanced chemiluminescence Western blotting substrate (Thermo), followed by visualization using an Amersham Imager 680 system (GE Healthcare, MA, USA). The level of β-actin was used as an internal control.
10
Immunoblot Analysis of Protein Expression
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