Cells were lysed into lysis buffer containing 1% Na deoxycholate (Sigma), 150mM NaCl, 10mM PO4Na2/K, pH 7.2 and supplemented with inhibitor cocktail (Sigma), 2mM EDTA, 1mM sodium orthovanadate (New England Biolabs), phosphatase inhibitor (Thermo Fisher) and 5mM PMSF (phenylmethanesulfonyl fluoride, Sigma). Benzonase nuclease (Santa Cruz) was added to reduce viscosity in protein extracts. Protein content was measured using BCA method (Biorad). Each sample was dissolved in Laemmli 5X buffer, and then denatured at 100°C for 10min. After SDS-PAGE and transfer onto nitrocellulose membrane (Hybond, GE Healthcare), membranes were incubated with goat polyclonal primary antibodies against p-AMPk (1/1000, Millipore) or AMPk (1/1000, Sigma) overnight at 4°C. Membranes were incubated with anti-goat peroxidase-conjugated anti-IgG secondary antibody (1/5000, Santa Cruz), developed using ECL substrate solution, exposed to the Fusion Fx7 (Thermo Fisher) and analyzed using Quantity One software (Biorad).
Fusion fx7
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The Fusion Fx7 is a compact and versatile laboratory instrument designed for fluorescence detection. It is capable of performing various fluorescence-based assays, including quantitative analysis and imaging. The Fusion Fx7 provides researchers with a reliable and efficient tool for their laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Benzonase nuclease, by Santa Cruz Biotechnology (1 mentions)
Sodium orthovanadate, by New England Biolabs (1 mentions)
Na deoxycholate, by Merck Group (1 mentions)
Bca method, by Bio-Rad (1 mentions)
Hybond, by Cytiva (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab79852, by Abcam (1 mentions)
Ab194583, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Ab8226, by Abcam (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
5 protocols using fusion fx7
1
Quantitative Analysis of AMPK Phosphorylation
2
Mitochondrial Protein Extraction and Western Blot Analysis
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Proteins from cells or lungs were extracted using RIPA Lysis Buffer with Complete Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). To detect the release of AIF from mitochondria, cells or tissue mitochondria isolation kit (Beyotime, Shanghai, China) were used to separate the protein, and cytoplasm (without mitochondria fractions) was collected for WB. WB was performed as described previously [23 (link)]. The protein levels were measured with specific primary antibodies including HSP70 (ab79852, Abcam, Cambridge, UK), KANK2 (PA5-116620, Invitrogen, Waltham, MA, USA), Bax (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab194583, Abcam, Cambridge, UK), GAPDH (9485, Abcam, Cambridge, UK), β-actin (ab8226, Abcam, Cambridge, UK), and AIF (4642, Cell Signaling, Danvers, MA, USA) at a dilution of 1:1000. The appropriate secondary antibodies were chosen to incubate with membrane for 2 h at room temperature. Finally, protein bands were applied with enhanced chemiluminescence detection kit (Thermo Scientific, Portsmouth, NH, USA) and imaged by fusion-capture software (Fusion FX7, Marne-la-Vallée, France).
3
Western Blotting of Autophagy and Apoptosis Proteins
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Cell lysates were prepared by RIPA buffer (1XPBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, US). After equal amounts of proteins were loaded to the gels, proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (EMD Millipore, Thermo Fisher Scientific, US). Following the classic immunoblotting steps (blocking, incubating with primary and secondary antibodies), chemiluminescence signals were detected using Clarity ECL substrate solution (Bio-Rad, US) by Fusion-FX7 (Vilber Lourmat, Thermo Fisher Scientific, US). Monoclonal antibodies used in this study were anti-LC3 (CST-12741, USA), anti-Atg-5/12 (CST-12994, USA), anti-Atg-7 (CST-8558, US), anti-actin (Sigma-Aldrich-A5316, UK), anti-caspase-3 (CST-9665, US), Anti-CHOP (CST-2895, UK). The experiments were repeated three times independently; with one representative result shown.
4
Immunoblotting Protocol for Protein Analysis
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Cells were rinsed with ice-cold PBS and lysed for 30 min at 4 °C in RIPA lysis and extraction buffer (#R0278, Sigma Aldrich, St.) supplemented with a protease/phosphatase inhibitor cocktail (#11697498001, Roche, Basel, Switzerland). Lysates were pelleted for 10 min at 13,000× g at 4 °C and supernatants were collected for protein quantitation (DC protein assay kit, Biorad, Hercules, CA, USA). After denaturation, 40 µg total proteins of each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (iBlot2, ThermoFisher Scientific, Waltham, MA, USA) for membrane blocking and immunoblotting with the primary antibody (anti-HA, #sc-805, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. After washing, blots were incubated for 1 h with a horseradish peroxidase-linked anti-rabbit antibody (Amersham, brand of GE Healthcare Europe GmbH, Velizy-Villacoublay, France) and processed for chemiluminescent substrate (Amersham ECL Select detection reagent kit, Sigma Aldrich, St.) according to the manufacturer’s instructions. Signal was detected using Fusion Fx7 (Thermo Fisher Scientific, Waltham, MA, USA) imaging system. β-actin (#A5316, Sigma Aldrich, St.) was used as a loading control.
5
Rac1 Activation in Intestinal Epithelium
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Intestinal epithelium was homogenized in 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% and the Fusion FX7 chemoluminescence System (Thermo Fisher Scientific, Illkirch, France). The membranes were then reprobed using a mouse mAb directed against total Rac1 (#05-389, Millipore). The autoradiographic signals were anayzed using the ImageJ software.
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