Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) analysis was performed as described previously13 (link). In brief, harvested cells resuspended in mitochondrial isolation buffer (Beyotime Biotechnology) were subjected to dounce-homogenization to lyse the cell. The homogenate was centrifuged at 700 × g for 10 min at 4 °C to spin out the cell debris and nucleus. The supernatant was further centrifuged at 10,000 × g for 30 min at 4 °C to pellet the intact crude mitochondria. Crude mitochondria (P5) were lysed in 1×sample buffer (0.5× TBE, 10% glycerol, 2% SDS and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). The proteins were transferred to an Immobilon membrane (Millipore) as mentioned above for further immunoblot analysis after electrophoresis in the running buffer (0.5× TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4 °C.
Vertical 1.5 agarose gel
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The Vertical 1.5% agarose gel is a laboratory instrument used for the separation and analysis of DNA, RNA, and protein molecules. It consists of a solidified agarose matrix with a 1.5% concentration, which serves as the medium for electrophoretic separation. The vertical orientation of the gel allows for efficient and uniform separation of the target molecules.
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10 protocols using vertical 1.5 agarose gel
1
Mitochondrial Protein Analysis by SDD-AGE
2
Mitochondrial Protein Separation by SDD-AGE
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Semidenaturing detergent agarose gel electrophoresis (SDD-AGE) was performed as previously described.14 (link) In brief, crude mitochondria were resuspended in 1× sample buffer (0.5× TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in running buffer (1×TBE and 0.01% SDS) for 50 min with a constant voltage of 100 V at 4 °C, the proteins were transferred to an immobile membrane (Millipore) for immunoblot analysis.
3
Mitochondrial Protein SDD-AGE Analysis
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SDD-AGE was performed as described before [14 (link), 43 (link)]. In brief, mitochondria were resuspended in 1x sample buffer (0.5× TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in the running buffer (1× TBE and 0.1% SDS) for 45 min with a constant voltage of 100 V at 4°C, the proteins were transferred to Immobilon membrane (Millipore) for immunoblot.
4
Crude Mitochondrial Fractionation and Analysis
5
Semi-denaturing Agarose Gel Electrophoresis
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Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was conducted according to the published protocol36 (link). Crude cell lysis was resuspended in 1×sample buffer (0.5×TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad, USA). After electrophoresis in the running buffer (1×TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4 °C, the proteins were transferred to Immobilon membrane (Millipore, USA) for immunoblotting and the following procedures are same as the Western-blot assay.
6
SDD-AGE Protein Extraction and Immunoblotting
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SDD-AGE was performed according to a published protocol with minor modifications70 (link). Cells were collected and lysed for 10 min with 200 μl lysis buffer (20 mM Tris–HCl, pH 7.4–7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing inhibitors for protease and phosphotases (Biotool). After centrifugation for 10 min at 14,000×g, supernatants were collected and added with 1× sample buffer (0.5× TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in the running buffer (1× TBE and 0.1% SDS) for 60 min with a constant voltage of 100 V at 4 °C, the proteins were subject to immunoblot analysis.
7
SDD-AGE Assay Protocol
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The SDD-AGE assay was conducted according to the established protocol (Hou et al., 2011 (link)). In short, crude cell lysis was resuspended in 1 × sample buffer (0.5 × TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad, United States). After electrophoresis in the running buffer (1 × TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4°C, the proteins were transferred to Immobilon membrane (Millipore, Billerica, Mass) for immunoblotting and the following procedures are same as the Western-blot assay.
8
Mitochondrial Protein Separation by SDD-AGE
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As described previously (32 (link)), mitochondria were isolated using the qProteome mitochondria isolation kit (Qiagen), and mitochondria pellets were suspended in sample buffer [0.5× tris-borate EDTA (TBE), 10% glycerol, 2% SDS, and 0.0025% bromophenol blue] and subjected to SDD-AGE. Samples were loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in the running buffer (1× TBE and 0.1% SDS) for 40 min with a constant voltage of 80 to 100 V at 4°C, the proteins were transferred to Immobilon membrane (Millipore) for immunoblotting.
9
SDD-AGE for Protein Separation
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SDD-AGE was performed as previously described [32 (link), 33 (link)]. In brief, cell lysates were resuspended in 1 × sample buffer (0.5 × TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in running buffer (1 × TBE and 0.01% SDS) for 50 min with a constant voltage 100 V at 4°C, the proteins were transferred to immobilon membrane (Millipore) for immunoblotting analysis.
10
Transfection and Viral Infection Immunoblotting
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HEK293T cells were transfected with the indicated plasmids for 24 h, and BMDCs were infected with SeV or EMCV for 6 h, then the cells were harvested and resuspended in buffer A (10 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.25 M d-mannitol) and lysed by grinding. Cell lysates were resuspended in 1×sample buffer (0.5×TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). The samples were electrophoresed in running buffer (1×TBE and 0.01%SDS) for 1h with a constant voltage 100 V at 4°C, the proteins were transferred to PVDF membranes (Millipore) for immunoblotting analysis.
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