Mitochondria were isolated from indicated C2C12 myoblasts and were lysed in BN lysis buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 2.5 mM MgCl2, 10% glycerol, 0.1 mM EDTA, 0.5 mM PMSF, 1.5% or 0.5% digitonin; AG scientific) at a concentration of 5 mg/ml for 30 min on ice. After a centrifugation at 21,000g for 10 min the mitochondrial extract was separated on a NativePAGE 3-12% Bis-Tris Gels (Invitrogen) or 4-15% Precast-Glgel 4-15% Native PAGE Gels (Sangon Biotech). Proteins were transferred to a PVDF membrane and detected by immunoblot using the indicated antibodies.
Nativepage 3 12 bis tris gel
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The NativePAGE™ 3–12% Bis-Tris gels are laboratory equipment designed for the separation and analysis of native protein complexes. These pre-cast polyacrylamide gels are suitable for running native protein electrophoresis to preserve the native structure and interactions of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
N dodecyl β d maltoside, by Merck Group (3 mentions)
Nativepage cathode buffer additive, by Thermo Fisher Scientific (3 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (3 mentions)
Precast glgel 4 15 native page gels, by Sangon (2 mentions)
Digitonin, by AG Scientific (2 mentions)
G 250 sample additive, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Native page buffer, by Thermo Fisher Scientific (2 mentions)
Native sample buffer, by Thermo Fisher Scientific (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Nativepage 5 g 250 sample additive, by Thermo Fisher Scientific (2 mentions)
Cytochrome c from equine heart, by Merck Group (2 mentions)
Catalase from bovine liver, by Merck Group (2 mentions)
Phenazine methosulfate, by Merck Group (2 mentions)
3 3 diaminobenzidine tetrahydrochloride hydrate, by Merck Group (2 mentions)
Axcell surelock mini cell vertical electrophoresis cell, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Imager 600, by GE Healthcare (1 mentions)
Goat anti mouse igg, by Bio-Rad (1 mentions)
Goat anti rabbit igg, by Bio-Rad (1 mentions)
31 protocols using nativepage 3 12 bis tris gel
1
Isolation and Analysis of Mitochondrial Complexes
2
Immunodetection of Phosphorylated Insulin Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Electrophoresis was performed with precast 4–12% Bis-Tris gels with MOPS SDS running buffer or 3–8% Tris-acetate gels or NativePAGE 3–12% Bis-Tris gels (Invitrogen) using the corresponding commercial running buffers (Invitrogen). Proteins were stained with Coomassie blue or silver stain or were transferred to polyvinylidene difluoride membranes for immunodetection. Membranes were blocked overnight with 5% (wt/vol) nonfat dry milk powder in TBST (0.1% [vol/vol] Tween-20 in 20 mM Tris, pH 7.4, and 150 mM NaCl) supplemented with phosphatase inhibitors (1 µM NaVO4 and 20 µM β-glycerophosphate). Membranes were probed with antibodies against phospho-IRβ (Tyr-1150/1151; 1:1,000; 3024; RRID, AB_331253; Cell Signaling Technology) or against total IRβ C terminus (1:2,000; 3020; RRID, AB_2249166; Cell Signaling Technology). The secondary antibodies goat anti–rabbit IgG (H+L; 170-6515; RRID, AB_11125142; Bio-Rad Laboratories) or goat anti–mouse IgG (H+L; 170-6516; RRID, AB_11125547; Bio-Rad Laboratories) conjugated to horseradish peroxidase were used at a 1:10,000 dilution, and detection was performed using SuperSignal West Femto maximum-sensitivity substrate electrochemiluminescence substrate (Thermo Fisher Scientific) with a charge-coupled device imager (Imager 600; GE Healthcare).
3
Isolation and Analysis of Mitochondrial Complexes
4
Native PAGE Analysis of Sg Enc-CLD-mNeonGreen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 1 μg of purified Sg Enc-CLD-mNeonGreen variants, were loaded onto Invitrogen Native PAGE 3–12% Bis-Tris gels. The native PAGE gels were run using 1x Invitrogen Native PAGE buffer for 120 min at 150 V at 4°C. In-gel mNeonGreen fluorescence was imaged using a GelDoc Go imaging system (BioRad Laboratories, Inc) equipped with a blue sample tray. Gels were then stained overnight using Ready Blue Stain (Sigma Aldrich), destained briefly in water, and imaged using a ChemiDoc Imaging system (BioRad Laboratories, Inc). Protein bands on Coomassie-stained and fluorescently imaged Native PAGE gels were analyzed via gel densitometry in ImageJ292 (link). Protein amount per lane was normalized to the Sg Enc-CLD-mNeonGreen sample. The same approach was taken to obtain a normalized mNeonGreen fluorescence intensities for each sample. Relative cargo-loading was then calculated as a ratio of normalized protein amount and normalized fluorescence intensiy.
5
Multidimensional Proteomic Analysis of Mitochondria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two hundred and fifty micrograms of isolated mitochondria were resuspended in 1× native sample buffer (Invitrogen) 2% digitonin and 1× protease inhibitors (Roche, Basel, Switzerland) and incubated for 1 h on ice, centrifuged at 20 000 g for 30 min at 4°C before the supernatant was removed and addition of 0.5% G-250 sample additive (Invitrogen). First-dimensional blue-native PAGE (BN-PAGE) was run on NativePAGE™ 3–12% Bis-Tris gels under native conditions at 150 V for 30 min with 1× native cathode buffer (Invitrogen) followed by 90 min at 250 V in 0.1× native cathode buffer. Second-dimensional BN-PAGE, first-dimensional lanes are cut from the gel and incubated in 1% SDS with 1% beta-mercaptoethanol for 1 h before the gel slice is loaded into a NuPAGE 4–13% 1.0 mm × two-dimensional (2D) gel (Invitrogen). Gels were run at 200 V for 50 min at room temperature in MOPS. Transfer for PVDF membrane and remaining steps were performed as with western blot.
6
Native PAGE Analysis of PvCPS
7
Native PAGE Analysis of RNA Pol II
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA Pol II was single-step purified from 4 mg of total protein obtained from 100 OD600 units of cells. Eluates were concentrated to 25 µl and analyzed by Native PAGE 3–12% Bis-Tris gels (Invitrogen). From total extracts 25 µg proteins were analyzed by Native PAGE. When indicated total extracts were treated either by 1 µg/ml RNaseA for 5 min at RT, or by 20 units/ml of DNase I (New England BioLabs Inc.) for 10 min at 37°C. After Native PAGE samples were analyzed by western blotting.
8
Assessing Mitochondrial Complex I Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fifty micrograms of isolated mitochondria were resuspended in 1× native sample buffer (Invitrogen) 2% digitonin and 1× protease inhibitors (Roche, Basel, Switzerland) and incubated for 1 h on ice before addition of 0.5% G-250 sample additive (Invitrogen). Prepared samples were run on NativePAGE™ 3–12% Bis-Tris gels under native conditions at 150 V for 30 min with 1× native cathode buffer (Invitrogen) followed by 90 min at 250 V in 0.1× native cathode buffer. The gel was then incubated for 1 h in 150 µM NADH, 3 mM nitro blue tetrazolium and 2 mM Tris-HCl (pH 7.4). Activity of Complex I corresponds to the depth of the blue-purple stain.
9
SDS-PAGE and BN-PAGE Analysis of Cytb6f Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For SDS–PAGE analysis of purified cytb6f, protein samples were mixed with an equal volume of 2× Laemmli sample buffer (Merck) and boiled for 10 min prior to separation on precast NuPAGE 12% Bis-Tris gels (Invitrogen). For BN-PAGE analysis, cytb6f was diluted in 4× sample buffer (100 mM Tris–HCl pH 7.5, 0.05% (w/v) bromphenol blue, 40% (w/v) glycerol) and analyzed on precast NativePAGE 3–12% Bis-Tris gels (Invitrogen). Gels were stained with Coomassie Brilliant Blue and imaged using an Amersham 600 imager (GE Healthcare). Alternatively, SDS–PAGE separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher Scientific) as described previously [37 (link)] and cytochrome c-mediated chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) and an Amersham Imager 600 (GE Healthcare).
10
Mitochondrial Protein Complex Separation by Blue-Native Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Blue-Native electrophoresis, proteins of isolated mitochondrial fractions (2 mg protein for immunoblotting with anti-F1b and 20 mg for immunoblotting with anti-Cyt1 antibodies) were incubated in 100 ml of Blue-Native PAGE buffer (50 mM NaCl, 50 mM imidazole, 1 mM EDTA, 3% digitonin, pH 7.0) for 10 min at 4 C (Wittig et al., 2006) and mixed with 13 ml of Native PAGE 5% G-250 Sample Additive (Invitrogen) and 18 ml of 30% glycerol. The samples were separated by electrophoresis on NativePAGE 3%-12% Bis-Tris gels (Invitrogen) with NativePAGE Anode Buffer and NativePAGE Dark Blue Cathode Buffer (Invitrogen) for 35 min at 150 V and then with NativePAGE Anode Buffer and NativePAGE Light Blue Cathode Buffer (Invitrogen) for 140 min at 250 V.
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!