Total proteins were extracted with cell lysis buffer (Sigma-Aldrich Inc.) in the presence of protease inhibitors (Halt™ Protease Inhibitor Cocktail (100×) and EDTA (100×), ThermoFisher Scientific) and quantified according to the BCA method (Pierce). Equal amounts (~3.8 µg) of protein/sample were separated on 4–20% gradient SDS-polyacrylamide gels (Bio-Rad Laboratories). Proteins were transferred to PVDF membranes (Bio-Rad Laboratories) and blocked with 5% bovine serum albumin (BSA) in PBS+ 0.05% Tween 20. Western blots were performed with mouse or rabbit antibodies for mitofusin-1 (1/2000, Abcam), mitofusin-2 (0.5 µg/ml, Abcam), DRP1 (1:500, Novus Biologicals), OPA1 (1:1000, Novus Biologicals), ATPB for the β subunit of the ATP synthase (0.5 µg/ml, Abcam) and VDAC1 (1 µg/ml, Abcam). To assure equal amount of loading, GAPDH (1 µg/ml, Abcam) was used as loading controls. Secondary antibodies were applied for 1 h and quantitative densitometry was performed after chemiluminescence detection using SuperSignal West Pico chemiluminescence substrate (Pierce) and corrections were made for background and loading control.
Mitofusin 2
Manufactured by Abcam
Sourced in United States
Mitofusin-2 is a protein that plays a role in mitochondrial fusion. It is involved in the process of merging two mitochondria to form a single, interconnected mitochondrial network.
Automatically generated - may contain errors
Lab products found in correlation
Vdac1, by Abcam (3 mentions)
Cell lysis buffer, by Merck Group (1 mentions)
Gradient sds polyacrylamide gel, by Bio-Rad (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Mitofusin 1, by Abcam (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Chemiluminescence substance, by Thermo Fisher Scientific (1 mentions)
Total drp1, by Cell Signaling Technology (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Actin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
Ecl protocol, by GE Healthcare (1 mentions)
Tomm20, by Merck Group (1 mentions)
Cox 1, by Santa Cruz Biotechnology (1 mentions)
7 protocols using mitofusin 2
1
Mitochondrial Dynamics Protein Analysis
2
Western Blotting of Mitochondrial Proteins
3
Mitochondrial Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared from 10 cryosections (20 μm thick) by means of Qiagen Tissue Lyser (Qiagen GmbH, Hilden, Germany) in a buffer containing 50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L DTT, 10% glycerol, 2% SDS, 1% Triton X‐100, Roche Complete Protease Inhibitor Cocktail (Roche Diagnostics S.p.a., Monza, MB, Italy), 1 mmol/L PMSF, 1 mmol/L NaVO3, 5 mmol/L NaF, and 3 mmol/L β‐glycerophosphate. Protein concentration was determined by the colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid method (Pierce™ BCA assay; Thermo Scientific, Rockford, IL), subsequently separated on 4–12% gradient SDS‐PAGE and electrotransfered onto nitrocellulose membrane which was then probed with different antibodies: MCU (product number HPA016480, 1:500; Sigma‐Aldrich), Actin (product number A2066, 1:15000; Sigma‐Aldrich); TOM20 (product number sc11415; 1:1000; Santa Cruz Biotechnology, Segrate, MI, Italy); SDH‐A (product number #11998, 1:500; Cell Signaling Technology; Euroclone, Pero, MI, Italy), COX IV (product number #4844, 1:1000; Cell Signaling Technology), OPA1 (product number 612606, 1:2000; BD Biosciences, Milano, Italy), and Mitofusin 2 (product number ab50838, 1:1000; AbCam, Cambridge, U.K.). Signals were visualized via chemiluminescence as described (Zampieri et al. 2001 ).
4
Protein Expression Analysis in Heart Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heart tissue was homogenized in a RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitor cocktail tablets (Roche Basel, Switzerland)) at 4 °C. After centrifuging for 30 min at 14,000× g, the supernatants were stored at −80 °C. For Western blot analysis, samples were boiled for 5 min in Laemmli sample buffer (5% SDS, 10% glycerol, 25 mM Tris-HCl pH: 6.8, 10 mM DTT, 0.01% bromophenol blue), and equal amounts of protein (30 μg) were separated on 8-15% SDS-polyacrylamide electrophoresis gels (SDS-PAGE). The relative amounts of each protein were determined with the following polyclonal or monoclonal antibodies: COX-1 (sc-1752 Santa Cruz Biotechnology, Dallas, TX, USA), COX-2 (160112Cayman Chemical Ann Arbor, Michigan USA); mitofusin 2 (ab56889Abcam Cambridge, UK,); TOMM20 (Sigma, WH0009804M1 Merck Life Science S.L.U. Darmstadt, Germany); β-actin (Sigma, A2066 Merck Life Science S.L.U. Darmstadt, Germany). After incubation with the corresponding horseradish peroxidase conjugated secondary antibody, blots were developed by the ECL protocol (GE Healthcare, Chalfont St Giles, UK). Protein band densities were normalized to β-actin. Densitometric analysis was carried out with Image J software and expressed in arbitrary units.
5
Western Blot Analysis of Autophagy and Mitochondrial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the mice organs were collected and disrupted in lysis buffer containing protease inhibitors (Roche Diagnostics, Berlin, Germany). After centrifugation, the supernatant was collected, and equivalent amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and an infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Western blot analyses were performed by ImageJ with anti-Gapdh (KM9002, Sungene, Tianjin, China), anti-Lc3b (SAB4200361, Sigma, St Louis, MO, USA), anti-Sqstm1 (PM045, MBL International, Japan), and anti-Eva1a (NB110-74787, Novusbio, Littleton, CO, USA) antibodies. Antibodies against Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 and against phosphorylated Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Drp1, Tomm20, Pink1, Parkin, Bnip3, Mitofusin2, and Pgc1 were purchased from Abcam (Cambridge, UK). DyLight 800/DyLight 680-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Rockland Immunochemicals (Limerick, PA, USA).
6
Mitochondrial Dysfunction and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gn-Rb1 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies against gp91phox, SOD2, 3-nitrotyrosine, 4 hydroxynonenal, DRP1, DRP1 (phospho S637), DRP1 (phospho S616), Mitofusin 2, OPA1, Fis1, Histone H3, caspases-3, cleaved caspases-3, and VDAC1 were obtained from Abcam (Cambridge, MA, USA). GAPDH, Bax, and Bcl-2 were obtained from Cell Signaling Technology (Beverly, MA, USA). Nrf2, keap1, NQO1, HO1, Ndufs1, Ndufv1, Ndufs6, Ndufs4, Ndufv2, and Ndufa12 were obtained from Abmart (Shanghai, China). Dihydroethidium (DHE) wasobtained from Beyotime (Jiangsu, China).
7
Pancreatic Tissue Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pancreatic slides were deparaffinized with Slidebrite (BioCare Medical, Concord CA) and dehydrated using graded ethanol concentrations. Slides were boiled in antigen retrieval citrate buffer pH6, blocked for 1hr with 2% normal goat serum, 2% bovine serum albumin, 0.5% Tween 20 in phosphate buffer saline followed by incubation with primary antibodies overnight at 4°C using different combinations. The staining series included antibodies to IP3R2 (Abcam, Cambridge, MA, USA 1:50), Translocase of outer mitochondrial membrane 20 (TOM20) (Santa Cruz biotechnologies, Dallas Texas, USA 1:50), VDAC-1 (Abcam, 1:100), mitofusin-2 (Abcam, 1:100), insulin (Dako, Carpinteria, CA, USA 1:150), glucagon (Abcam, 1:200). Secondary antibodies coupled to a fluorochrome (AF488, AF555, AF647, Life Technologies, Grand Island, NY, USA) were added for 30 min at RT according to the species of primary antibodies. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) and preparations were mounted in Prolong Gold anti-fade reagent (Life Technologies). Slides were analyzed with a slide scanner AxioScan.Z1 (Carl Zeiss SAS, Marly le Roi, France) at x40 magnification.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!