Samples were prepared using the NativePAGETM sample preparation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, purified recombinant CERT samples and HMW Native Marker Kit (GE Healthcare, Chicago, IL, USA) were diluted in native PAGE buffer supplemented with 1% digitonin, and CBB G-250 was added to a final concentration of 0.25%. Samples were separated on a NativePAGE™ Novex® 4–16% Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA), and the gel was incubated in 25 mM Tris-glycine buffer (pH 8.3) with 0.1% SDS for 15 min at room temperature with shaking. Proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA), and the membrane was fixed in 10% acetic acid for 15 min, rinsed with distilled water, and air-dried. The membrane was de-stained in methanol, rinsed with distilled water, and subjected to western blotting analysis.
Nativepage novex 4 16 bis tris gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NativePAGE™ Novex® 4-16% Bis-Tris gels are pre-cast polyacrylamide gels designed for the separation and analysis of native proteins under non-denaturing conditions. They feature a gradient of 4-16% polyacrylamide for the resolution of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Nativemark unstained protein standard, by Thermo Fisher Scientific (2 mentions)
Hmw native marker kit, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Nativepage novex bis tris gel system, by Thermo Fisher Scientific (1 mentions)
Nativepage 5 g 250 sample additive, by Thermo Fisher Scientific (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
Digitonin, by Thermo Fisher Scientific (1 mentions)
Dark blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Light blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Coomassie blue g 250, by Thermo Fisher Scientific (1 mentions)
Mitochondrial isolation kit, by Thermo Fisher Scientific (1 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
Goat anti apoe antibody, by Meridian Bioscience (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Cocktail of protease inhibitor, by Roche (1 mentions)
10 protocols using nativepage novex 4 16 bis tris gel
1
Native PAGE and Western Blot Analysis of CERT Protein
2
Mitochondrial Protein Complex Analysis
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Suspended mitochondria were added to an equal volume of 2× NativePAGE Sample Buffer (Thermo Fisher Scientific) containing 2% (w/v) digitonin, and left on ice for 30 min. After centrifugation (11 500g at 4°C for 15 min), the supernatant was applied to a NativePAGE Novex 4–16% Bis-Tris Gel (Thermo Fisher Scientific) for electrophoresis following the manufacturer's instructions.
3
Protein Complex Analysis by Blue Native-PAGE
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Blue native-PAGE was performed using the Native PAGE Novex bis-Tris Gel System (Invitrogen). Five micrograms of protein analytes were mixed with NativePAGE sample buffer (Invitrogen) and NativePAGE 5% G-250 sample additive (Invitrogen). Mixtures were incubated for 30 min on ice. After incubation, analytes were applied to NativePAGE Novex 4–16% bis-Tris gel (Invitrogen), and electrophoresis was performed at 150 V. Preparation of running buffer and other conditions on Blue Native-PAGE were carried out in accordance with the manufacturer’s instructions. NativeMark unstained protein standard (Invitrogen) was used as a molecular weight marker for Blue Native-PAGE. Gels were stained with CBB R-250.
4
Isolation and Analysis of Protein Complexes
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BN-PAGE analysis was performed as described previously (Kikuchi et al., 2006) . Protoplasts were resuspended in solubilization buffer (50 mM Bis-Tris-HCl [pH 7.0], 0.5 M aminocaproic acid, 10% w/v glycerol, 2% n-Dodecyl b-D-maltoside, and 1% protease inhibitor cocktail). The resuspended pellets were incubated on ice for 10 min and centrifuged at 20 000 g. Insoluble materials were removed by ultracentrifugation at 100 000 g for 10 min. The supernatant was combined with Coomassie brilliant blue G-250, and the samples were loaded onto a 4%-16% gradient gel (Native PAGE Novex 4%-16% Bis-Tris Gel; Invitrogen, Carlsbad, CA, USA). The cathode tank buffer contained 50 mM Tricine/15 mM Bis-Tris (pH 7.0), and 0.02% CBB-G-250, and the anode tank buffer contained 50 mM Bis-Tris (pH 7.0). Gel electrophoresis was performed at 4 C. Western blot analysis was performed using anti-HA antibody.
5
Protein Separation using SDS-PAGE and BN-PAGE
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SDS–PAGE of the extracted proteins was performed using NuPAGE 4–12% acrylamide Bis-tris gels (ThermoFisher, Carlsbad, CA). BN-PAGE of the extracted proteins was performed using NativePAGE Novex 4–16% Bis-Tris gels (ThermoFisher). A native PAGE unstained protein standard (ThermoFisher) was used as soluble protein standard. A membrane protein standard was prepared by following the protocol of Wittig et al. (Wittig et al. 2010 (link)).
6
Protein Separation in Synechococcus 7002
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Whole-cell extracts of Synechococcus 7002 were prepared as described previously [75 (link)] and proteins were separated by SDS-PAGE [76 (link)] or BN-PAGE using precast NativePAGE Novex 4–16% Bis–Tris gels (Thermofisher, Germany). Preparation of thylakoid membranes for BN-PAGE analysis (Additional file 10 ) and gel electrophoresis was performed according to Lassen et al. [77 (link)].
7
Solubilization and Native PAGE Separation of Membrane Protein Complexes
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Approximately 10 µg of B-fraction was solubilized in 30 µl of 1.25% digitonin (Invitrogen) by incubation for 15 min at room temperature with intermittent vortexing. After solubilization, the samples were centrifuged at 16,000 x g for 20 min at 20˚C to pellet the insoluble material. The supernatant (20.6 µl) was mixed with Native PAGE 4X Sample Buffer (7.5 µl) (Invitrogen) and 5% (w/v) Coomassie blue G-250 (1.86 μl) (Invitrogen) just prior to loading samples onto the Blue Native gel.
NativePAGE™ Novex® 4-16% Bis-Tris gels were used according to the manufacturer´s instructions (Invitrogen). The first third of the run was performed with Dark Blue Cathode Buffer (Invitrogen), and the remaining two-thirds were performed with Light Blue cathode Buffer (Invitrogen). The Native Mark Unstained (Invitrogen) was used as the molecular mass standard.
NativePAGE™ Novex® 4-16% Bis-Tris gels were used according to the manufacturer´s instructions (Invitrogen). The first third of the run was performed with Dark Blue Cathode Buffer (Invitrogen), and the remaining two-thirds were performed with Light Blue cathode Buffer (Invitrogen). The Native Mark Unstained (Invitrogen) was used as the molecular mass standard.
8
Romo1 Oligomerization in Mitochondria
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Mitochondria were isolated from HEK293 cells using a mitochondrial isolation kit (Thermo Fisher Scientific). To assess Romo1 oligomerization, mitochondria isolated from HEK293 cells were lysed with 0.5% digitonin or 1% ODG for 1 h on ice. Lysates were analyzed by BN-PAGE using NativePAGE Novex 4–16% Bis-Tris gels (Invitrogen). A BN-PAGE gel strip was used for secondary SDS-PAGE. For the homooligomerization assay, synthesized Romo1 was incubated with 50 mM DHPC, 300 mM sucrose, and 10 mM Hepes/Tris, pH 7, or added to MIM-LUVs in 300 mM sucrose and 10 mM Hepes/Tris, pH 7, for 2 h at room temperature. For MIM-LUV–reconstituted Romo1, samples were lysed with 1% ODG before BN/SDS-PAGE.
9
Quantifying ApoE and APP Levels
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Cortical tissues were homogenized with a Polytron homogenizer in ice cold TBS buffer containing protease inhibitor and phosphate inhibitor cocktails (Roche). Proteins were separated by Native PAGE™ Novex 4–16% Bis-Tris gels (Invitrogen) under native conditions following the manufacturer’s instructions and transferred to PVDF (Millipore) at 100 V for 1 h using the Trans-Blot Cell (Bio-Rad). Blots were treated with Ponceau S Staining Solution (0.1% (w/v) Ponceau S in 5% (v/v) acetic acid) to visualize the molecular mass markers. The NativeMark Unstained Protein Standard from Invitrogen was used for estimation of particle sizes. After washing in TBS, blots were processed for Western blot. The membrane was incubated with goat anti-apoE antibody (K74180B, Meridian Life Science) overnight at 4°C, followed by peroxidase-labeled donkey anti-goat antibody (Santa Cruz). The membrane was developed with Lumigen ECL Ultra Western Blotting HRP Substrate (Lumigen), and the signals were detected by Fuji film Luminescent Image Analyzer (LAS4000). An antibody that recognizes the C-terminus of APP (18961, IBL-America) was used for detecting APP and its C-terminal fragments. Anti-β-actin (Sigma) was used as a loading control. Western blot bands were quantified by Image J software.
10
Blue Native Gel Electrophoresis of Sperm Proteins
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Capacitated spermatozoa were prepared for Blue Native polyacrylamide gel electrophoresis (BN-PAGE) as previously described (Redgrove et al., 2013) . Briefly, cell pellets were resuspended in native lysis buffer consisting of 1% n-dodecyl b-D-maltoside, 0.5% Coomassie Blue G250 and a cocktail of protease inhibitors (Roche, Mannheim, Germany) and incubated at 48C on an orbital rocker for 30 min. Samples were centrifuged for 20 min at 14 000g and then dialysed against Blue Native cathode buffer (purchased from Invitrogen, Carlsbad, CA, USA) overnight at 48C. Dialysed native protein lysates were loaded onto blue native polyacrylamide gels (Native-PAGE Novex 4 -16%, Bis-Tris gels; Invitrogen) and resolved using a Native-PAGE cathode and anode buffer (Redgrove et al., 2011) and run at 48C at 100 V for the duration of the stacking gel layer and at 200 V for the resolving gel layer for 3 h. Following retrieval from the cassettes, gels were either stained with Coomassie G250 or prepared for western blotting.
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