Some of the rescued homozygous oz2 mutants (oz2/oz2/OZ*) were able to produce a reasonable amount of viable seeds. Mitochondria were extracted from liquid-grown Arabidopsis seedlings following the protocol of Murcha and Whelan (26 (link)). The yield of mitochondrial extraction was evaluated by Bradford assay (Bio-Rad). The sample preparation for the Blue native (BN) gel was done according to the protocol of Schertl and Braun (27 (link)). The gel used for the BN-PAGE was a pre-cast Native PAGE Novex 3–12% Bis–Tris gel (Invitrogen). The conditions for the run were: 4°C, 150 V for 60 min, 250 V for 180 min. Approximately 50 μg of mitochondrial protein was loaded per lane. After completion of the run, slices of the gel were either stained with Coomassie R-250 according to the manufacturer's instructions, or incubated overnight with freshly prepared solutions for in gel-activity at room temperature following the protocol of Schertl and Braun (27 (link)). The reactions were then stopped by transferring the gels into fixing solution.
Native page novex 3 12 bis tris gel
Manufactured by Thermo Fisher Scientific
The Native PAGE Novex 3–12% Bis–Tris gel is a pre-cast polyacrylamide gel used for the separation and analysis of native proteins under non-denaturing conditions. It is designed to maintain the native structure and biological activity of proteins during electrophoresis.
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Lab products found in correlation
Bradford assay, by Bio-Rad (1 mentions)
Native page buffer, by Thermo Fisher Scientific (1 mentions)
N dodecyl β d maltoside, by Thermo Fisher Scientific (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
N dodecyl β d maltoside, by Dojindo Laboratories (1 mentions)
Nativepage novex bis tris gel system, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
5 protocols using native page novex 3 12 bis tris gel
1
Isolation and Activity Analysis of Arabidopsis Mitochondria
2
Shikonin-Induced BN Lysate Preparation
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After 48h of Shikonin treatment, BN lysates were prepared from UM-UC3 cells in 20 mM Bis-Tris (pH7.4), 125 mM caproic acid, 20 mM KCl, 2mM EDTA, 5 mM MgCl2, 10% glycerol and 2% n-dodecyl beta-D-maltoside (DDM) followed by three freezing and thawing cycles and centrifugation at 14,000 g for 30 min at 4°C. Protein concentration was determined as described above and equal amounts of protein loaded on a Native PAGE Novex 3–12% Bis-tris gel (Invitrogen, Carlsbad, CA) and electrophoresed according to manufacturer’s instructions.
3
Native PAGE for mitochondrial complexes
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High-resolution clear native PAGE and activity staining were performed as described.29 (link) In brief, isolated mitochondria were solubilized with Native PAGE Sample Buffer (Invitrogen) containing 0.3% n-dodecyl-β-d -maltoside (Dojindo, Kumamoto, Japan). Thirty micrograms of protein was applied to NativePAGE Novex 3%–12% Bis-Tris Gel (Invitrogen). Native PAGE Buffer (Invitrogen) was used as the anode buffer, and Native PAGE Buffer containing 0.02% n-dodecyl-β-d -maltoside and 0.05% deoxycholate was used as the cathode buffer.
For in-gel catalytic activity assays, the gels were incubated as described.29 (link)
For in-gel catalytic activity assays, the gels were incubated as described.29 (link)
4
Native PAGE Immunoblotting for Protein Complexes
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Blue native PAGE was performed according to the instructions for the NativePAGE Novex Bis-Tris Gel System (Invitrogen), in which addition of detergent (digitonin) is required to avoid protein aggregation. Livers were homogenized in 4× NativePAGE Sample Buffer (50 μM Bis Tris, 0.1% Glycerol, 50 μM NaCl, and 0.00001% Ponceau S) and 1% digitonin. After the lysates had been centrifuged at 15,000 rpm, 5% G-250 was added to the supernatants which were then electrophoresed using NativePAGE Novex3-12% Bis-Tris Gel (Invitrogen). The gels were then incubated in 0.1% SDS and transferred to a PVDF membrane (Millipore). After blocking in PBS containing 0.1% Tween 20 and 3% skim milk, the membrane was incubated with the appropriate primary antibody, followed by incubation with ECL anti-rabbit/mouse IgG Horseradish Peroxidase linked whole antibody (GE Healthcare).
5
Native Detection of Procollagen and Collagen
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For the detection of type-I procollagen and collagen in the conditioned medium the use of antibody that recognized the protein in its native soluble form (non-denatured) was needed in order to avoid the detection of collagen peptides from the product used as supplement in the culture medium. Test was done to assure no positive signal for the testproduct. Just prior to loading samples (30 mg/lane) onto precast polyacrylamide gel (NativePAGE Novex 3-12% Bis-Tris Gel, Invitrogen), 2.5 mL of NativePAGE 5% G-250 sample additive was added to samples on ice. Then, the native gel electrophoresis was run according to manufacturer's instructions. Proteins were transferred to a PVDF membrane which was soaked in the blocking buffer (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4, 0.1% Tween-20 containing 5% skimmed milk) for 1 h. The primary mouse monoclonal antibody against type-I procollagen (QED Bioscience Inc.) and type I collagen (Sigma-Aldrich) were used at a dilution of 1:500 in blocking buffer. Secondary antimouse monoclonal antibodies conjugated with horseradish peroxidase (Amersham Inc., Arlington Heights, IL, USA) were diluted to 1:2000 in blocking buffer. The resulting bands were detected by the horseradish peroxidase reaction with an enhanced chemiluminescence kit (Amersham) and exposed to X-ray film (GE Technology).
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