Whole cell lysates were prepared using RIPA lysis buffer (Millipore), with complete protease inhibitors (Roche, Basel, Switzerland). After estimating the protein concentration with BCA Protein Assay Kit (Thermo Fisher), 10–50 μg (per lane) of the cell lysates or co-immunoprecipitation (co-IP) products were subjected to SDS-PAGE and then blotted onto a polyvinylidene difluoride membrane (Millipore). The HRP conjugated anti-rabbit IgG (1:10000, Sigma-Aldrich) or anti-mouse IgG (1:10000, Sigma-Aldrich) were used to reveal antibody binding. Immunoreactive complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposed to a GE Amersham Imager 600 machine.
Amersham imager 600 machine
Manufactured by GE Healthcare
Sourced in United States
The Amersham Imager 600 is a compact, automated instrument designed for imaging and analyzing a wide range of gel-based and membrane-based samples, including Western blots, Northern blots, and colony/plaque assays. The system utilizes a charge-coupled device (CCD) camera and a selection of imaging modules to capture high-quality images of the samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg, by Merck Group (1 mentions)
Anti flag monoclonal antibody, by Merck Group (1 mentions)
Anti myc tag antibody, by Proteintech (1 mentions)
Complete protease inhibitor, by Beyotime (1 mentions)
Hrp conjugated anti mouse igg, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Antibody against β actin, by Beyotime (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Na934 1ml, by GE Healthcare (1 mentions)
Nxa931 1ml, by GE Healthcare (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
6 protocols using amersham imager 600 machine
1
Western Blot Analysis of Protein Complexes
2
Antibody Detection in Whole-Cell Lysates
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Antibodies were obtained from the following companies: monoclonal anti-FLAG antibody (F1804, Sigma-Aldrich), anti-MYC-Tag antibody (66004-I-Ig, Proteintech, Chicago, IL, USA), and anti-Nectin-2 antibody (ab135246, Abcam, Cambridge, UK). Whole-cell lysates were prepared using RIPA lysis buffer (Millipore), with complete protease inhibitors (Roche, Basel, Switzerland). The BCA Protein Assay Kit (Thermo Fisher) was used to determine the protein concentration. The HRP-conjugated anti-rabbit IgG (1:10000, Sigma-Aldrich) or anti-mouse IgG (1:10000, Sigma-Aldrich) were used to reveal antibody binding. Immunoreactive complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposed to a GE Amersham Imager 600 machine.
3
Western Blot Analysis of Immune Mediators
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Whole-cell lysates were obtained using RIPA lysis buffer containing complete protease inhibitors (Beyotime Biotechnology). The protein concentration was assayed using a BCA Protein Assay Kit (Beyotime Biotechnology). The antibody against mouse IRF5 (1:1,000; CST), antibody against β-actin (1:1,000; Beyotime Biotechnology), antibody against IL-1β (1:1,000; CST), antibody against IL-6 (1:1,000; Santa Cruz), antibody against IL-12 (1:1,000; Santa Cruz), antibody against TNFα (1:1,000; CST), antibody against mouse TRAF6 (1:1,000; Abcam), horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin G (IgG) (1:5,000; Beyotime Biotechnology), and HRP-conjugated antimouse IgG (1:5,000; Beyotime Biotechnology) were used to detect the expression of IRF5, IL-1β, IL-6, IL-12, TNFα, TRAF6, and β-actin. The signals were detected using the Immobilon Western Chemiluminescent HRP Substrate (Wanleibio) and analyzed using the GE Amersham Imager 600 machine.
4
Western Blot Analysis of ARID2, AKT, and GAPDH
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Xenograft tissues and HCC cells were lysed with RIPA lysis buffer (Beyotime, Beijing, China) on ice. A Enhanced BCA Protein Assay kit (Beyotime) was used for measuring protein concentration. The protein samples were separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes then were incubated at 4°C overnight with specific primary antibody against ARID2 (ab166850, Abcam, Cambridge, UK), p-AKT (Ser473, #4060, Cell Signaling Technology, Beverly, MA, USA) , AKT (#4691, Cell Signaling Technology) and GAPDH (sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (NXA931-1ML and NA934-1ML, GE Healthcare Life Sciences, Beijing, China) at room temperature for 2 h. The blots were visualized by Chemiluminescence Reagents (Sigma, St-Louis, MO, USA) and pictures were captured by Amersham™ Imager 600 machine (GE Healthcare Life Sciences).
5
Protein Expression Analysis of 5-FU Treated Cells
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AB1‐HA cells were treated or untreated with IC50 of 5‐FU (1 μM) for 24 h; 48 h or 72 h at 37°C, followed by homogenization in M‐PER reagents (Thermo Fisher) containing phosphatase and protease inhibitor cocktails (Roche). Protein concentrations were determined using a TaKaRa Bradford Protein Assay kit (Takara) and read at 450 nm with a SUNRISE Remote R microplate reader (Tecan). The protein lysates were transferred to polyvinylidene fluoride membrane and blocked by blocking one solution for 1 h (Nacalai Tesque) The membranes were incubated overnight at 4°C with primary antibodies (dilution: 1:1000) to S100A8 (Cell Signaling Technology), Bv8 (Abcam) and β‐actin (Santa Cruz Biotechnology). After washing, membranes were applied using horseradish peroxidase (HRP)‐conjugated secondary antibodies (GE Healthcare), and visualized through a chemiluminescent Amersham Imager 600 machine.
6
Protein Expression Analysis by Western Blot
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Total proteins were extracted by RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) supplemented with protease (Thermo Fisher Scientific) and phosphatase inhibitors (Roche). Protein concentrations were measured by the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of total protein were resolved on a 10% polyacrylamide gel and then transferred to a polyvinyl difluoride (PVDF) membrane. After blocking with 5% milk or 5% BSA for 1 h at room temperature, the PVDF membranes were incubated overnight at 4°C on a shaking table with antibodies against FKBP51, IGFBP1, PRL, vimentin, CK8+18, p-S473 AKT, p-T308 AKT, total AKT and FOXO1A (Abcam) and GAPDH (Santa Cruz). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Santa Cruz) for 1 h and then detected using the Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore) and the Amersham Imager 600 machine (GE Healthcare). The exposure time of the membrane was automatically detected by the machine. The grey values of the strips were analysed by ImageJ. GAPDH was used as an endogenous control for normalization. Applications of the antibodies are shown in Table 1. All Western blots were performed at least three times from independent experiments, and representative results are shown.
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