Protein samples were mixed with sample buffer (250 mM Tris-HCl, at pH 6.8, 10% sodium dodecylsulfate (SDS), 30% Glycerol, 5% β-mercaptoethanol, and 0.02% bromophenol blue) and boiled for 5 min. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, which was followed by incubation with the primary antibody overnight at 4 °C. After washing with PBST (PBS and 0.25% Tween-20), the membrane was incubated with a secondary antibody conjugated with horseradish peroxide (Millipore) for 1 h. The membrane was washed with PBST and detected using an enhanced chemiluminescence (ECL) reagent kit (Millipore) followed by exposure to Amersham Imager 600 imagers (GE, Pittsburgh, PA, USA). ImageJ software (http://rsb.info.nih.gov/ij/index.html , NIH, Bethesda, MA, USA) was used to analyze the relative quantification of the ECL signals.
Amersham imager 600 imager
Manufactured by GE Healthcare
Sourced in United States
The Amersham Imager 600 is a versatile imaging system designed for a wide range of applications in life science research. It utilizes a high-resolution CCD camera and advanced imaging technology to capture and analyze a variety of samples, including Western blots, gels, and chemiluminescent and fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ecl chromogenic kit, by Thermo Fisher Scientific (2 mentions)
Ab6789, by Abcam (2 mentions)
P ampkα, by Cell Signaling Technology (2 mentions)
Ampkα, by Cell Signaling Technology (2 mentions)
Gel pro analyzer 4, by Meyer Instruments (2 mentions)
Complete edta free, by Roche (2 mentions)
Ecl reagent kit, by Merck Group (1 mentions)
Criterion tgx precast gel, by Bio-Rad (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Primary antibody dilution buffer, by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Quick start bradford 1 dye reagent, by Bio-Rad (1 mentions)
Mouse cytokine antibody array, by Abcam (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
16 protocols using amersham imager 600 imager
1
Western Blot Protein Analysis
2
Influenza Virus Protein Detection
3
Western Blot Analysis of Breast Cancer Cell Line
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Breast cancer cell line MDA-MB-231 (1 × 106 cells/each well in 6-well plates) was transfected with NC, miR-29b-3p mimics and miR-29b-3p inhibitor for 48 h. Cell lysates were collected using 1 mL RIPA (Solarbio, Beijing, China) with 10 μL PMSF. Protein concentration of cell lysates were measured with a BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). Western blot was carried out by separating 40 µg of protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (240 mA, 4 h). The membranes were blocked with 5% non-fat milk and washed with TBST buffer and then incubated with primary antibody Cleaved PARP (Asp214) (D64E10), NF-κB p65 (D14E12), phospho-NF-κB p65 (ser536), IκB-α (44D4), phospho-IκB-α (14D4), (Cell Signaling Technology, USA) at a dilution of 1:1000 in Primary Antibody Dilution Buffer (Beyotime. Shanghai, China) at 4 °C overnight. Next, membrane treated with primary antibody were washed 3 times with TBST and incubated with an anti-rabbit HRP-linked antibody at a 1:5000 dilutions for 1 h at room temperature, β-Actin was used as an internal reference. The signals were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) with the Amersham Imager 600 imagers (GE healthcare Life science, Pittsburgh, USA).
4
Anlotinib Dose-Response Assay in GIST Cell Lines
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Anlotinib with 5 concentrations, 0, 10, 50, 200, and 500 nmol/L, was added to the 2 GIST cell lines for 4 h when the confluence of the cells were 90%. The whole cell lysates were prepared using a standard protocol. The protein lysate concentrations were measured using a Quick Start Bradford 1 × Dye Reagent (Bio-Rad; #5000205). Electrophoresis and western blotting were performed using standard techniques. The hybridization signals were detected by chemiluminescence (Immobilon Western, Millipore Corporation, MA) and captured using an Amersham Imager 600 imagers (GE Healthcare; #29083461).
5
Western Blot Analysis of Recombinant Proteins
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The cell lysates or purified recombinant proteins were subjected to SDS-PAGE and transferred to NC membrane using a Pyxis™ Gel Processor (Pyxis, China). The NC membrane was blocked in 5% BSA and then incubated in anti-MBP antibodies at a 1:8000 dilution, or anti-serum against IpaJ at a 1:1000 dilution for 2 h at 37 °C. After incubation with goat anti-mouse IgG-HRP at a 1:10000 dilution for 1 h, the NC membrane was stained with an ECL chromogenic kit (Thermo, USA) and scanned by using an Amersham Imager 600 imagers (GE Healthcare, USA).
6
Profiling Intracellular Cytokines in Frozen Cells
7
Western Blot Analysis of Recombinant Proteins
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The cell lysates or purified recombinant proteins were subjected to SDS-PAGE on a 12% polyacrylamide gel in Tris-glycine running buffer (pH 8.7), and transferred to PVDF membrane (Pall, USA) by PyxisTM Gel Processor (Pyxis, China). The PVDF membrane was blocked in 5% BSA and then incubated with anti-MBP antibody or anti-IpaJ MAbs in blocking buffer for 2 h at 37°C. After washing, the membranes were incubated with goat anti-mouse IgG-HRP (Sigma, USA) for 1 h. The PVDF membrane was then stained with the ECL chromogenic kit (Thermo, USA) and scanned using the Amersham Imager 600 imagers (GE Healthcare, USA).
8
Western Blot Analysis of Adipose Tissue
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Total protein of brown adipose tissue (BAT), subcutaneous white adipose tissues (sWAT), and epididymal white adipose tissues (eWAT) were lysed by RIPA buffer, and quantitated by Pierce TM BCA Protein Assay kit (23225, ThermoFisher Scienti c). Equal amounts of protein (20 μg) were separated by SDS-PAGE electrophoresis, transferred to PVDF membrane, and blocked in 5% skim milk in TBST. Then, the membrane was incubated overnight with primary antibody solution at 4 ºC. After rinsing the blot, the membrane was incubated in the horseradish peroxidase (HRP)-conjugated secondary antibody solution for 2 h at room temperature. Finally, chemiluminescent signals on the blot were applied with SuperSignal™ West Femto Maximum Sensitivity Substrate (34095, ThermoFisher Scienti c), and captured using Amersham Imager 600 imagers (GE Healthcare Life Sciences). The band intensity of protein was read by image analysis software Fiji. The following primary and secondary antibodies were used in this study: Ago2 (RN003M, MBL International, 1:1000), UCP1 (#14670, Cell Signaling Technology, 1:1000), AMPKα (#2532, Cell Signaling Technology, 1:1000), p-AMPKα (#2535, Cell Signaling Technology, 1:1000), GAPDH (ab8245, Abcam, 1:1000), Goat Anti-Rabbit IgG H&L (HRP) (ab6721, Abcam, 1:10000), Goat Anti-Mouse IgG H&L (HRP) (ab6789, Abcam, 1:10000).
9
Western Blot Analysis of Adipose Tissue
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Total protein of brown adipose tissue (BAT), subcutaneous white adipose tissues (sWAT), and epididymal white adipose tissues (eWAT) were lysed by RIPA buffer, and quantitated by Pierce TM BCA Protein Assay kit (23225, ThermoFisher Scienti c). Equal amounts of protein (20 μg) were separated by SDS-PAGE electrophoresis, transferred to PVDF membrane, and blocked in 5% skim milk in TBST. Then, the membrane was incubated overnight with primary antibody solution at 4 ºC. After rinsing the blot, the membrane was incubated in the horseradish peroxidase (HRP)-conjugated secondary antibody solution for 2 h at room temperature. Finally, chemiluminescent signals on the blot were applied with SuperSignal™ West Femto Maximum Sensitivity Substrate (34095, ThermoFisher Scienti c), and captured using Amersham Imager 600 imagers (GE Healthcare Life Sciences). The band intensity of protein was read by image analysis software Fiji. The following primary and secondary antibodies were used in this study: Ago2 (RN003M, MBL International, 1:1000), UCP1 (#14670, Cell Signaling Technology, 1:1000), AMPKα (#2532, Cell Signaling Technology, 1:1000), p-AMPKα (#2535, Cell Signaling Technology, 1:1000), GAPDH (ab8245, Abcam, 1:1000), Goat Anti-Rabbit IgG H&L (HRP) (ab6721, Abcam, 1:10000), Goat Anti-Mouse IgG H&L (HRP) (ab6789, Abcam, 1:10000).
10
Comprehensive Western Blot and Immunoprecipitation Workflow
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For Western blots, cells were pelleted and lysed directly with denaturing gel loading buffer (Tris-HCl 125 mM, pH 6.8, glycerol 20% vol/vol, SDS 4% vol/vol, β-mercaptoethanol 10% vol/vol, and bromophenol blue). The primary and secondary antibodies are described in Table S6 . Western blots were developed by chemiluminescence (Pierce Super Signal West Dura; Thermo Fisher Scientific) and imaged using an Amersham Imager 600 imager (GE Healthcare Life Sciences). Bands were quantified with Gel-Pro Analyzer 4 (Meyer Instruments).
For immunoprecipitations, cells were serum starved for 48 h, and proteins were extracted with lysis buffer (20 mM Hepes, pH 7.5, 50 mM KCl, and 1 mM MgCl2) with 0.5% digitonin and protease inhibitor (cOmplete EDTA-Free; Roche). Insoluble components were removed by centrifugation at 20,000 g. Primary antibodies preadsorbed to protein G-sepharose beads (GE Healthcare) were added to the cell extract, and the mixture was incubated for 2 h at 4°C. After centrifugation, beads were washed with lysis buffer supplemented with 0.1% digitonin before elution in denaturing gel loading buffer for SDS-PAGE electrophoresis and Western blot analysis.
Isolation of mRNA and quantitative mRNA analysis was performed as previously described (Jonassen et al., 2008 (link)) using the primers tabulated inTable S7 .
For immunoprecipitations, cells were serum starved for 48 h, and proteins were extracted with lysis buffer (20 mM Hepes, pH 7.5, 50 mM KCl, and 1 mM MgCl2) with 0.5% digitonin and protease inhibitor (cOmplete EDTA-Free; Roche). Insoluble components were removed by centrifugation at 20,000 g. Primary antibodies preadsorbed to protein G-sepharose beads (GE Healthcare) were added to the cell extract, and the mixture was incubated for 2 h at 4°C. After centrifugation, beads were washed with lysis buffer supplemented with 0.1% digitonin before elution in denaturing gel loading buffer for SDS-PAGE electrophoresis and Western blot analysis.
Isolation of mRNA and quantitative mRNA analysis was performed as previously described (Jonassen et al., 2008 (link)) using the primers tabulated in
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