Proteins were extracted and separated as described previously (Baier et al., 2018 (link)) using 10% SDS‐Polyacrylamide gels, followed by colloidal Coomassie staining (Dyballa and Metzger, 2009 (link)) and Western blotting onto a nitrocellulose membrane (AmershamTM; GE Healthcare, Uppsala, Sweden). After blocking with 2.5% (w/v) BSA and 2.5% (w/v) milk powder in TBS‐T buffer, immunodetection was carried out via incubation with horseradish peroxidase coupled Strep II tag (IBA Life Sciences, Göttingen, Germany) or HA‐tag antibodies (Thermo Scientific) in blocking buffer (1:10000), washing with TBS‐T and addition of Pierce™ ECL Western blotting substrates (Thermo Scientific), followed by detection in a Fusion Fx7 chemiluminescence imaging system (Vilber Lourmat).
Fusion fx7 chemiluminescence imaging system
Manufactured by Vilber
Sourced in France, United Kingdom
The Fusion FX7 is a chemiluminescence imaging system designed for capturing and analyzing luminescent signals. It features a high-resolution CMOS camera, adjustable lighting, and a temperature-controlled sample area. The system is capable of detecting a wide range of chemiluminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti mouse immunoglobulin g horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Hrp substrate, by Cytiva (1 mentions)
Anti fgf21 antibody, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by GenDEPOT (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ecl prime solution, by Cytiva (1 mentions)
Cw0049, by CWBIO (1 mentions)
Se250, by Hoefer (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Tissue protein extraction kit, by CWBIO (1 mentions)
6 protocols using fusion fx7 chemiluminescence imaging system
1
Protein Extraction and Analysis Protocol
2
Immunoblotting of Pluripotency Markers
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Western blot technique was conducted as described previously (Akgun et al., 2019). The membranes were probed sequentially with SMAD family member 4 (SMAD4) (#38454), sex determing region Y HMG-box 2 (SOX2) (#3579), octamer-binding transcription factor (OCT4) (#2750), epidermal growth factor receptor (EGFR) (#4267) and GAPDH (#2118) antibodies (Cell Signaling Technology, MA, USA). Then, HRP-linked anti-rabbit IgG antibodies (Cell Signaling Technologies, MA, USA) were used. HRP bound to membranes were visualized with the Fusion FX-7 chemiluminescence imaging system (Vilber Lourmat, Torcy, France). The experiment was repeated two independently experiment.
3
Quantifying KISS1R Expression in HEK293 Cells
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FLAG-KISS1R-HEK293 cells were scraped into HEPES buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2, and 0.25 M sucrose) and homogenized on ice. The homogenate was centrifuged at 800g at 4°C for 20 minutes and the supernatant was centrifuged twice at 100,000g at 4°C for 1 hour. The cell pellet was solubilized with Cell-LyEX MP (TOYO B-Net, Tokyo, Japan). A 1-μg aliquot of this fraction with urea (final concentration, 5 M) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis [10 (link)] and transferred to a polyvinylidene fluoride membrane. The transfer membrane was incubated with anti-FLAG M2 antibody (1:1000 dilution) overnight at 4°C, followed by incubation with anti-mouse immunoglobulin G horseradish-peroxidase-linked antibody (RRID: AB_330924; 1:10,000 dilution; Cell Signaling Technology, Danvers, MA) for 1 hour at room temperature. The immunoreactive band was visualized with ImmunoStar LD (Wako Pure Chemicals, Osaka, Japan) and analyzed using a Fusion FX7 Chemiluminescence Imaging System (Vilber-Lourmat, Eberhardzell, Germany). To calculate relative KISS1R expression, Na,K-ATPase (RRID: AB_2060983; 1:1000 dilution; Cell Signaling Technology) was used as a loading control.
4
Western Blot Analysis of FGF21 in WATs
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Total protein from WATs was extracted using a tissue homogenizer and lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1% 4-nonylphenyl-polyethylene glycol, 5 mM ethylenediaminetetraacetic acid, 1 M threo-1,4-Dimercapto-2,3-butanediol) containing protease inhibitor cocktail (GenDEPOT, Barker, TX, USA). The homogenates were centrifuged for 15 min at 15,928 g and 4°C. The supernatants were collected and centrifuged again, and the second supernatants were used for Western blotting analysis. To detect mouse FGF21 protein, protein-loaded membranes were first reacted with anti-FGF21 antibody (diluted 1:1000, Abcam, Cambridge, MA, USA) and then incubated with HRP-conjugated secondary antibody (diluted 1:5000, Thermo Scientific, Rockford, IL, USA). Finally, the FGF21 protein was detected with HRP substrate (Amersham Bioscience, Buckinghamshire, UK) using a Fusion FX7 chemiluminescence imaging system (Vilber Lourmat, France).
5
Western Blot Analysis of Cellular Signaling
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The cells were lysed in RIPA containing phosphatase and protease inhibitor cocktail, and the protein concentrations were evaluated using a BCA protein assay kit (Thermo Scientific, IL, USA). Equal amounts of the total proteins were subjected to a 10% SDS-Polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk in phosphate-buffered saline (PBS) at RT for 1 h, the membranes were incubated with the primary antibodies such as iNOS (sc-650), COX-2 (sc-376861), HO-1 (sc-10789), Nrf2 (sc-722), p-IκB (#9246), IκB (#9242), p-NF-κB (#3037), Lamin B (sc-374015), β-actin (sc-47778), p-ERK1/2 (sc-7383), p-JNK (#9251), p-p38 MAPK (#9211), ERK1/2 (sc-94), JNK (sc-7345), and p38 MAPK(sc-535) at 4 °C overnight, followed by incubation with the proper secondary antibodies conjugated with horseradish peroxidase at RT for 1 h. The blots were developed using ECL prime solution (Amersham Bioscience, Buckinghamshire, UK), and visualized using a Fusion FX7 chemiluminescence imaging system (Vilber Lourmat, Marne-la-vallée, France).
6
Hippocampal Protein Extraction and Western Blot
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Total protein was extracted from small pieces of hippocampus tissue using Tissue Protein Extraction kit (CW0891, CWBiotech). Protein concentration was determined by BCA Protein Assay kit (CW0014, CWBiotech). All protein samples were separated by 12% separation gel electrophoresis (Hoefer, SE250, USA) at 120 V for 150 min. Then, the resolved protein was transferred onto PVDF membrane (Millipore) using wet-transfer method (Hoefer, TE250, USA) at 240 mA for 150 min. The membrane was washed with Tris buffered saline with 1% Tween-20 (TBST) and then blocked with 5% BSA (CW0049, CWBiotech) for 2 h at 37°C. It was then incubated with anti-Aβ 1-42 and anti-β-actin antibodies overnight at 4°C. Afterwards, the membranes were washed and incubated with goat anti-rabbit IgG conjugated to HRP secondary antibody for 40 min at 37°C. Immunoreactive bands were detected using an enhanced chemiluminescent kit (ECL, CW0049, CWBiotech). The blots were imaged using Fusion FX7 Chemiluminescence imaging system (Vilber, France) and quantifications were performed using the Image J v.1.44 software and normalized based on the intensity of the β-actin band.
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