Isozyme electrophoresis was carried out following the methods of Esbenshade and Triantaphyllou (1985) [29 (link)] and Esbenshade and Triantaphyllou (1990) [30 (link)]. Phenotypes were observed for esterases (Est) and malate dehydrogenase (Mdh). Five young egg-laying females of M. vitis sp. nov. and five young egg-laying females from a previously identified population of M. javanica (used for comparison) were prepared and placed in microtubes containing 10 μL mixed liquor consisting of 20% sucrose, 2% Triton X-100 and 0.02% bromophenol blue, the nematodes were broken with a sterile dissecting needle and the enzyme solution can be used immediately or stored at -20°C refrigerator until use. Electrophoresis was carried out in separating and stacking gels consisted of 7% and 3% polyacrylamide, respectively, 0.75 mm thick, with Tris-glycine buffer (PH8.7) in a Mini-PROTEAN® Tetra Cell apparatus (Bio-Rad). Voltage was maintained at 80 volts for the first 30 minutes, the following was maintained at 150 volts of the separation period until the bromophenol blue dye had migrated to approximately 0.5 cm ahead of the bottom of the gel. Gels was stained with Mdh stain solution for Mdh and with Est stain solution for Est, the preparation of Mdh and Est stain solution following the method of Esbenshade and Triantaphyllou (1985) [31 ].
Mini protean tetra cell apparatus
Manufactured by Bio-Rad
Sourced in United States, Austria
The Mini-PROTEAN Tetra Cell apparatus is a vertical gel electrophoresis system designed for the separation and analysis of proteins. It provides a compact and efficient platform for performing SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) experiments.
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Lab products found in correlation
Pageruler prestained ladder, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Immobilon polyvinyl difluoride membrane, by Merck Group (1 mentions)
Mini trans blot module, by Bio-Rad (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Gstrap ff column, by GE Healthcare (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Ecl western blotting detection kit, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Ab6160, by Abcam (1 mentions)
Storage phospho screen, by GE Healthcare (1 mentions)
Typhoon 9410 scanner, by GE Healthcare (1 mentions)
22 protocols using mini protean tetra cell apparatus
1
Isozyme Electrophoresis for Meloidogyne Identification
2
SDS-PAGE and Western Blot Analysis of Bacterial Strains
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SDS-PAGE and western blot analysis were carried out to check the reactivity of Sal-06mAb with different bacterial strains belonging to Enterobacteriaceae. Briefly, SDS-PAGE was performed using Bio-Rad Mini-PROTEAN Tetra Cell apparatus as per the procedure of Laemmli (1970 (link)) with minor modifications. One milliliter of overnight bacterial cells was harvested by centrifugation and dissolved in equal volumes of 1× PBS and 2× Laemmli buffer. The bacterial samples were subjected to boiling for 10 min followed by centrifugation at 13,500g for 5 min to separate cell debris. Ten microliters of cell lysate supernatant was loaded on 12% SDS-PAGE gels and resolved at 100 V. Separated proteins on resolved gels were transferred to charged nitrocellulose membrane (0.45 μm, Pall Life Sciences) by electroblotting by wet transfer in Bio-Rad Mini Trans-Blot cell at 70 V for 60 min. After blotting, membranes were blocked in 5% skim milk solution overnight at 4 °C. Excess milk protein was washed in PBST (Tween 20, 0.05%) solution. Next, the membrane was incubated in 1:2000 dilutions of Sal-06mAb at room temperature on a gel rocker. The membrane was washed in PBST and incubated with goat anti-mouse IgG conjugated with HRP (1:5000 dilutions). After washing the membrane with PBST, the blots were developed using diaminobenzidine tetrahydrochloride hydrate and 0.003% H2O2 solution in PBS.
3
Western Blot Analysis of Protein Signaling
4
Purification and Characterization of GST-SlCaM6 Fusion Protein
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pDEST15 vector expressing GST‐SlCaM6 was transformed into Rosetta™(DE3) competent cells, and the fusion protein was induced and purified with GSTrap column (GSTrap FF column, GE Healthcare) following the user manual. Native polyacrylamide gel electrophoresis (Native‐PAGE) was carried out as described previously (Niepmann & Zheng, 2006 ; Arndt et al., 2012 ). Equal volumes of 133 μM synthetic peptide and 50 μM GST‐ or His‐ SlCaM6 were mixed and incubated for 1 h at 4°C. Peptide–protein interactions were analysed on 12% tris‐glycine native gels (30 mM Tris‐HCl pH 7.5, 190 mM glycine, 5 mM DTT). NativeMark Unstained Protein Standard (Life Technologies) was used as a protein marker for the analysis. The electrophoresis was performed with the Mini‐PROTEAN Tetra Cell apparatus (BioRad) in native running buffer (25 mM Tris base, 192 mM Glycine) at a low current (10 mA) at 4°C. The proteins on the gel were visualized by Coomassie blue staining (Blakesley & Boezi, 1977 ).
5
Isolation and Purification of Bacterial LPS
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LPS was isolated by the hot phenol–water method [38 ]. Briefly, freeze-dried bacteria were suspended in 45% phenol solution (2 g/50 ml) and incubated at 65 °C with intermittent stirring for 15 min. The suspension was cooled to 5 °C, centrifuged (3500 g, 30 min), and the aqueous phase was collected. Water was added to compensate for the collected aqueous phase volume, and the cycle was repeated. The aqueous phases were combined and dialyzed against deionized water to remove residual phenol, then filtered and freeze-dried. The obtained crude LPS was dissolved in MiliQ water and purified by ultracentrifugation (105,000 g, 6 h, 4 °C) [39 (link)]. LPS extracts were analyzed by SDS-PAGE using the Laemmli buffer system [40 (link)]. Electrophoresis was performed using 6% polyacrylamide stacking gels and 15% separating gels. Samples were loaded into the gels after mixing with Laemmli buffer and heating at 98 °C for 4 min. The SDS-PAGE separation of LPS was performed at constant voltage (120 V), for 90 min using a Mini-Protean Tetra Cell apparatus (Bio-Rad). The separated LPS was visualized using silver staining according to Tsai and Frasch [41 (link)] with the modification of Fomsgaard [42 (link)] and imaged under white light using a GelDoc XR system (Bio-Rad).
6
Caco-2 Cell Protein Extraction and Western Blot
7
SDS-PAGE Analysis of HRP Isoenzymes
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To check the electrophoretic purity of HRP isoenzyme preparations SDS–PAGE was performed using a 5% stacking gel and a 10% separating gel in 1× Tris–glycine buffer. Unless otherwise stated, samples were diluted to a protein concentration between 0.1 and 0.5 mg mL−1 before loading. Gels were run in a vertical electrophoresis Mini-PROTEAN Tetra Cell apparatus (Biorad, Austria) and stained with Coomassie blue. The protein mass standard used was the PageRuler Prestained Ladder (Fermentas, Austria).
8
SDS-PAGE Analysis of Protein Samples
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Electrophoresis was done with aliquots of supernatants obtained at the end of the cultivation. SDS-PAGE was performed using a 6% stacking gel and a 12% separating gel in 1x Tris-glycine buffer. Gels were run in the vertical electrophoresis Mini-PROTEAN Tetra Cell apparatus (Biorad; Austria) at 150 V for about 2 h. Gels were stained with Coomassie blue. The protein mass standard used was the PageRuler Prestained Ladder (Fermentas; Austria).
9
Electrophoretic Mobility Shift Assay for DNA-Protein Binding
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DNA probes used for EMSA were prepared by PCR amplifying the desired sequences, using specific primer pairs (Supplementary Data 4 ) followed by purification of linear DNA from agarose gels using a gel extraction kit (Qiagen). EMSA was set up in the 15 µl reaction mixture containing 17 nM DNA and purified ResR/McdR in binding buffer (10 mM Tris-HCl, pH 8.0, 25 mM NaCl, 50 mM KCl, 10 mM MgCl2, 1 mM DTT and 0.002% dextran sulfate) for 15 minutes at 37 °C. Sample containing DNA without ResR/McdR was simultaneously used as control. The DNA-protein complexes were resolved in a native 6 % polyacrylamide gel (acrylamide: bisacrylamide, 28:1, w/w) in 0.5× Tris/Borate/EDTA buffer at room temperature for 2.5 hours at 65 V using the Mini-PROTEAN Tetra cell apparatus (Bio-Rad). After staining the gel with ethidium bromide, signals were visualized under the UV transilluminator. Each binding experiment was performed in duplicate to ascertain the binding of ResR/McdR with DNA.
10
Protein Separation and Visualization
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To obtain 100 μg of proteins for electrophoretic separation, the solubilized proteins were re-precipitated with cold ammonium acetate-saturated methanol. The precipitated proteins were then dissolved in Laemlli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 0.005% β-mercaptoethanol) and denatured by boiling at 95°C for 4 min [3 (link)]. 100 μg protein was loaded into each lane on a 1.0 mm in-house casted 12% polyacrylamide gel. Electrophoresis was conducted in a Bio-Rad mini-PROTEAN Tetra Cell apparatus (Bio-Rad Laboratories Inc., Hercules, CA) at 200 V for 1 h. Following electrophoresis, the separated proteins were fixed for 30 min in a fixing solution (50% ethanol, 10% acetic acid) and stained with an in-house prepared Colloidal Coomassie G-250. The gel was destained with Milli-Q water until the gel background was clear. The gel was scanned as digital image using Bio-5000 Plus scanner (Microtek, Hsinchu, Taiwan) according to the manufacturer’s instructions.
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