SDS-PAGE gels (7.5%) were prepared according to a standard protocol (Laemmli 1970 (link)). Crude cell extracts of T. forsythia ATCC 43037, UB4 and defined mutants thereof were run in a Mini Protean electrophoresis apparatus (Bio-Rad, Vienna, Austria) and proteins were visualized with colloidal CBB R-250. For Western immunoblot analyses, proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) using a Mini Trans-Blot Cell (Bio-Rad). Polyclonal antisera raised in rabbits against the recombinant S-layer proteins TfsA (α-TfsA) and TfsB (α-TfsB) (Sekot et al. 2012 (link)) were used as primary antibodies followed by a monoclonal goat anti-rabbit secondary antibody labeled with IRDye 800CW (LI-COR Biosciences, Lincoln, NE). S-layer protein bands were visualized at 800 nm using an Odyssey Infrared Imaging System (LI-COR Biosciences).
Mini protean electrophoresis apparatus
Manufactured by Bio-Rad
Sourced in United States, Austria
The Mini-PROTEAN electrophoresis apparatus is a compact and versatile system designed for performing polyacrylamide gel electrophoresis (PAGE) experiments. Its core function is to separate biomolecules, such as proteins or nucleic acids, based on their size and charge.
Automatically generated - may contain errors
Lab products found in correlation
Mini trans blot cell, by Bio-Rad (3 mentions)
Irdye 800cw, by LI COR (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Molecular standard marker, by Thermo Fisher Scientific (2 mentions)
Gel doc xr, by Bio-Rad (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
2 d quant kit, by GE Healthcare (2 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Semi dry transfer device, by GE Healthcare (1 mentions)
Western blot imager, by Vilber (1 mentions)
13 protocols using mini protean electrophoresis apparatus
1
SDS-PAGE and Western Blot Analysis of T. forsythia
2
PAGE Analysis of Recombinant PDE5 Isoforms
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PAGE was performed with a 5% nondenaturing acrylamide gel in a Tris–glycine pH 8.3 running buffer at 4 °C for 60–80 min with a current of 20 mA in a Bio-Rad Mini-Protean electrophoresis apparatus [38 ]. Each well was loaded with 1.0 μg of purified recombinant MmPDE5A1, MmPDE5A2 or MmPDE5A3 and was preincubated at 30 °C with substrate and/or inhibitor/modifier at the concentrations and times specified in the figures; the activity buffer consisted of 5 μL of 50 mM HEPES pH 7.5, 50 mM NaCl and 15 mM MgCl2. Protein bands were visualized through Coomassie staining. The concentrations of sildenafil and cGMP were higher than therapeutically used concentrations in order to demonstrate the conformational structural changes according to [19 (link)].
3
SDS-PAGE and Western Blot Analysis of T. forsythia S-layer Proteins
4
SDS-PAGE Analysis of Membrane Proteins
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SDS-PAGE was performed following the method of Li [34 (link)] with some modifications. Briefly, 1 mg/mL of MP samples (30 μL) from 2.6 was mixed with 10 μL 4 × DTT SDS-PAGE loading buffer. Then, the mixture was heated at 100 °C for 10 min. The loading mixture (10 μL) and molecular standard marker (5 μL, Thermo Fisher Scientific Co., Ltd., Shanghai, China) were loaded onto every precast gel lane (GenScript, 12% polyacrylamide, 15 wells). A MiniProtean electrophoresis apparatus (Bio-Rad Laboratories, Hercules, CA, USA) was used at 4 °C with the following voltage: 80 V for 20 min and 100 V for 80 min. Thereafter, the gel was stained for 40 min and decolored for 10 h by hand using a staining solution and decolorizing liquid (GenScript Biotech Corp, Nanjing, China). The gels were scanned using a molecular imaging system (Gel Doc XR+, Bio-Rad Laboratories, Hercules, CA, USA).
5
SDS-PAGE and Western Blot Analysis of T. forsythia
6
Quantitative Analysis of T. forsythia Proteins
7
SDS-PAGE Protein Separation and Visualization
8
SDS-PAGE Protein Separation and Visualization
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The gel contained a separating gel (7.5%) at the bottom and a stacking gel at the top. The samples were prepared by mixing 10 μL of protein solution (1 mg/mL) with 10 μL of loading buffer (2% SDS, 10% beta-mercaptoethanol (BME)) and heated in an 80 °C water bath for 5 min. Then, 15 μL of each sample was loaded in each well (7.5 μg of protein/well). The gel was run at 200 mV for 30 min with a Mini Protean Electrophoresis apparatus (Bio-Rad, Hercules, CA). Then, the gel was stained with stain I (10% v/v acetic acid, 10% v/v isopropanol, 0.003% m/v brilliant blue R-250) and stain II (20% v/v acetic acid, 0.003% m/v brilliant blue R-250) and destained with 10% v/v acetic-acid solution. The gels were scanned on an office scanner and digitized with NIH ImageJ software (V 1.52i., November, 2018, NIH, Bethesday, MA, USA).
9
Identification of Informative SNPs by SNP-PCR
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For SNP-PCR, DNA samples from twenty donor/recipient pairs were tested for these 18 SNPs to identify the informative SNP region.
The lists of primers for amplification of autosomal chromosomal SNPs were as follows:
The PCR was carried out in a 25 μl reaction mixture containing 5pM of each primer, 0.5 units of Taq polymerase (Fermentas, Lithuania), 30 mM of each dNTP (Fermentas Life sciences Lithuania), 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 mg/ml gelatin (Sigma, UK), and 0.2 μg of genomic DNA. Thermal cycling was done in 9700 (Perkin Elmer, USA) using 28 cycles of: Denaturation 940C for 30 seconds, Annealing 63°C for 30 seconds, and extension 72°C for one minute. The final extension was done for 3 minutes. The amplified products were loaded on 6% non-denaturing polyacrylamide gels measuring 1 mm x 10 cm x 10 cm on Mini-protean electrophoresis apparatus (Bio-Rad, USA). Electrophoresis was carried out at 150V for 40 minutes. The gels were stained by silver nitrate.
The lists of primers for amplification of autosomal chromosomal SNPs were as follows:
The PCR was carried out in a 25 μl reaction mixture containing 5pM of each primer, 0.5 units of Taq polymerase (Fermentas, Lithuania), 30 mM of each dNTP (Fermentas Life sciences Lithuania), 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 mg/ml gelatin (Sigma, UK), and 0.2 μg of genomic DNA. Thermal cycling was done in 9700 (Perkin Elmer, USA) using 28 cycles of: Denaturation 940C for 30 seconds, Annealing 63°C for 30 seconds, and extension 72°C for one minute. The final extension was done for 3 minutes. The amplified products were loaded on 6% non-denaturing polyacrylamide gels measuring 1 mm x 10 cm x 10 cm on Mini-protean electrophoresis apparatus (Bio-Rad, USA). Electrophoresis was carried out at 150V for 40 minutes. The gels were stained by silver nitrate.
10
SDS-PAGE Protein Denaturation and Separation
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SDS-PAGE procedures were carried out according to the method presented in Li [23 (link)]. The original 2.3.1 MP solution was utilized and adjusted to the concentration of 1 mg/mL, mixed with a 30 μL protein sample containing 10 μL 4×DTT SDS-PAGE sample buffer to create a final sampling buffer. This sample loading buffer was heated in a water bath at 100 °C for 10 min to denature proteins. A volume of 10 μL of the sample loading buffer then contained 5 μL molecular standard marker (Thermo Fisher Scientific Co., Ltd., Shanghai, China) and 5 μL of 4–20% precast gel lane (GenScript, 12% polyacrylamide, 15 wells). A MiniProtean electrophoresis apparatus (Bio-Rad Laboratories, Hercules, CA, USA) was used at 4 °C with the following voltage: 80 V for 20 min and 100 V for 80 min. Thereafter, the gel was stained for 40 min and decolored for 10 h by hand using a staining solution and decolorizing liquid (GenScript Biotech Corp, Nanjing, China). The gels were scanned using a molecular imaging system (Gel Doc XR+, Bio-Rad Laboratories, Hercules, CA, USA).
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