After 24 h of treatment, total protein was isolated from cells by RIPA lysate with protease inhibitor and phosphatase inhibitor. After determining the protein concentration by the BCA protein assay kit, 20 μg protein was added into each well of the vertical electrophoresis tank and separated by 10% SDS-PAGE. Subsequently, the protein was transferred onto the PVDF membrane. The Power Pac basic power supply, Wide mini-sub cell GT system, Trans-Blot SD Semi-Dry electrophoretic transfer tank were all provided by Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After being blocked by 5% skim-milk for 2 h, then the incubation of primary antibodies (TNF-α, p65, p-p65, IκBα, p-IκBα, GBP2, IRF1, SAMHD1, p-SAMHD1, β-actin, GAPDH, 1:2000) was performed overnight at 4 °C. After washing by TBST three times for 30 min, the secondary antibodies (anti-rabbit IgG HRP-linked antibody, 1:2000) were incubated for 1 h at room temperature. After TBST wash, the ECL chromogenic substrate was used to detect specific bands in Image Quant LAS 500 (GE HealthCare Technologies Inc., Chicago, IL, USA). The gray value was estimated by Image J software (National Institutes of Health, Bethesda, MD, USA) normalized to β-actin.
Powerpac basic power supply
Manufactured by Bio-Rad
Sourced in United States
The PowerPac Basic Power Supply is an essential laboratory equipment for the application of electrical currents in various biomedical and analytical techniques. It provides a stable and consistent power supply for the operation of electrophoresis, blotting, and other specialized laboratory instruments.
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Lab products found in correlation
Mini protean tetra cell, by Bio-Rad (4 mentions)
Mini trans blot module, by Bio-Rad (2 mentions)
Mini sub cell gt cell, by Bio-Rad (2 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (2 mentions)
Wide mini sub cell gt system, by Bio-Rad (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Readyblue protein gel stain, by Merck Group (1 mentions)
High molecular weight range gel filtration calibration kit, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
Broad multi color pre stained protein standard, by GenScript (1 mentions)
Expressplus page gel, by GenScript (1 mentions)
Mj mini gradient thermal cycler, by Bio-Rad (1 mentions)
Odyssey 9120 imaging system, by LI COR (1 mentions)
β actin, by Wuhan Servicebio Technology (1 mentions)
Tubulin, by Affinity Biosciences (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Gelstain, by Transgene (1 mentions)
Imagequant 300 imager, by GE Healthcare (1 mentions)
Power taq pcr mastermix, by BioTeke (1 mentions)
Pvdf transfer membrane, by Thermo Fisher Scientific (1 mentions)
25 protocols using powerpac basic power supply
1
Western Blot Analysis of Protein Expression
2
Native Protein Separation and Oligomeric State Determination
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The hrCN PAGE protocol was adapted from Lemaire et al. (2018) (link). Glycerol was added to the sample at a final amount of 20% (v/v). Ponceau S at a final concentration of 0.001% (w/v) served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM tricine, 15 mM Bis-Tris/HCl, pH 7.0, 0.05% (w/v) sodium deoxycholate, 2 mM DTT, and 0.01% (w/v) dodecyl maltoside, whilst the anode buffer contained 50 mM Bis-Tris/HCl, PH 7.0, 2 mM DTT. An 8–15% linear polyacrylamide gradient gel was used, and electrophoresis was run under a N2/CO2 (90:10%) atmosphere with a constant 40 mA current (PowerPac™ Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue™ Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark™ Unstained Protein Standard (Thermo Fischer Scientific, Driesch, Germany).
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N2/H2 (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N2/H2 (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.
3
SDS-PAGE Analysis of Protein Purity
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To assess purity, a polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions was performed, in a Mini-PROTEAN Tetra Cell (Biorad), mixing samples with sample buffer and loading 20 µL of samples and 5 µL of the Broad Multi Color Pre-Stained Protein Standard (Genscript) into the wells of ExpressPlus PAGE Gels (Genscript) and using Tris-MOPS-SDS buffer (Genscript) as running buffer, in a two-step running starting at 60V for 30 min and then changed to 110 V for 1 hour and 30 minutes, with a PowerPac Basic power supply (Biorad). Gels were stained with 0.25% Coomassie Blue R-250 for 4 hours and distained with a solution containing 10% acetic acid, 30% methanol, and 60% water, applying four washes of 30 min each. Both staining and distaining were performed using a rocking platform settled at 14 oscillations/min.
4
Multiplex PCR Assay for Virulence Genes in P. multocida
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A total of nine genes associated with virulence in P. multocida (sodA, ompH, tbpA, tadD, hsf1, hgbB, pfhA, ptfA, and toxA) were investigated. Three multiplex PCR reactions were established to detect the virulence factors sodA, ompH, and tbpA (reaction 1), virulence factors tadD, hsf1, and hgbB (reaction 2), and virulence factors pfhA and ptfA (reaction 3). The toxA gene was detected by simplex PCR reaction as previously described [18 (link)]. The PCR conditions, adapted from Gharib Mombeni et al. [37 (link)], were as follows: initial denaturation at 94 °C for 15 min; 25 cycles at 94 °C for 30 s, at 53 °C for 30 s, and at 72 °C for 60 s; with a final extension at 72 °C for 10 min. All reactions were performed using an MJ Mini Gradient Thermal Cycler (Bio-Rad, England). Simplex Taq PCR and Multiplex PCR Master Mix kits from Qiagen (Germantown, USA) were used in the reactions. PCR-generated products were detected by electrophoresis in 2.0% agarose gels supplemented with 1X SYBR safe DNA gel stain (Invitrogen SA) using a PowerPac basic power supply (Bio-Rad) at 90 V and 400 mA for 45 min. P. multocida strains NCTC 10322 (cap-A, tadD, pfhA, ptfA), NCTC 10323 (cap-B, tbpA, hgbB), NCTC 12178 (cap-D, lps-6, ompH, toxA), NCTC 10326 (cap-E, lps-2, sodA), and C104013 (cap-F, lps-3, hsf-1) were used as positive controls for the genes indicated.
5
Western Blot Protein Analysis Protocol
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Samples were mixed with 4X Laemmli buffer and boiled at 95°C for 5 min. 10% polyacrylamide gels were hand-casted and loaded with 20 μg of sample. The gels were run using a Mini-PROTEAN Tetra Cell (BioRad #1658001) and PowerPac Basic Power Supply (BioRad #1645050) at 100V for approximately 90 min. Gels were prepared for transfer to PVDF membranes, and the transfer was run using a Mini Trans-Blot Module (BioRad #1703935) at 400 mV for 60 min. The membranes were removed and blocked in 5% milk dissolved in TBST for 1 h. They were then incubated overnight with primary antibody [anti-tau (1:500,000), anti-GAPDH (1:1,000), and anti-Histone H3 (1:1,000)] overnight at 4°C on a shaker. The membranes were washed and incubated with secondary antibody [anti-rabbit AlexaFluor600 (1:5,000) and anti-mouse AlexaFluor800 (1:10,000)] for 1 h at room temperature. Imaging was done using a Licor Odyssey 9120 Imaging System. Quantification was done using ImageJ.
6
Quantitative NASH Protein Analysis
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Liver tissues from wild-type (wt) and NASH model mice were finely minced and then subjected to protein extraction via the RIPA method. The expression levels of β-actin and tubulin were normalized using their grayscale values measured by ImageJ. Polyacrylamide gels were prepared using the One-Step PAGE Gel Fast Preparation Kit (15%) from Vazyme (catalog number E305), with the 180 kDa Prestained Protein Marker from Vazyme (catalog number MP102) used for molecular weight estimation. Electrophoresis and membrane transfer were conducted using the PowerPac Basic Power Supply from BIO-RAD. Blocking was performed with 5% BSA. Primary antibodies were diluted as follows: β-actin at 1:1000 from Servicebio (catalog number GB15001-100), tubulin at 1:5000 from Affinity Biosciences (catalog number T0023), CDKN1B at 1:1000 from BIOSS (catalog number bs-0742R), and TFAM at 1:1000 from Proteintech (catalog number 22586-1-AP). Imaging was done using the Tanon 4800 system. Grayscale values for all bands were acquired with ImageJ, and the relative protein expression levels were determined using β-actin and tubulin as standards. Statistical analysis and graphical representation were performed using GraphPad Prism 9.
7
Salmonella Identification via invA and hilA
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The sequences of the primer pair targeting the invA gene used for the identification of Salmonella contamination were as follows: 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′ and 5′-TCATCGCACCGTCAAAGGAACC-3′ (Rahn et al., 1992 ). For confirmation, all environmental samples were screened using primers targeting hilA: 5′-GTGAAATTATCGCCACGTTCGGGCAA-3′ and 5′-TCATCGCACCGTCAAAGGAACC-3′ (Craciunas et al., 2012 ). Amplification was carried out in a 20 µL reaction mixture (10 µL 2 × Power Taq PCR MasterMix [BioTeke Corporation, Beijing, China], 1 µL of 10 µM of each of the primers, 2 µL of DNA template, and 6 µL of ddH2O) in an Applied Biosystems Veriti 96-well thermal cycler (Life Technologies, CA, USA). The thermal cycles were: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, and final extension at 72°C for 10 min.
Genomic DNA of Salmonella strains and ddH2O were systematically included as positive and negative controls in each assay, respectively. Five-microliter aliquots of PCR product were analyzed by electrophoresis on 2% (w/v) agarose gel (BIOWEST, Nuaillé, France) using a PowerPac Basic Power Supply (Bio-Rad Laboratories, CA, USA). Gels were stained with GelStain (TransGen Biotech, Beijing, China) and visualized under UV light using an ImageQuant 300 Imager (GE Healthcare, Buckinghamshire, UK).
Genomic DNA of Salmonella strains and ddH2O were systematically included as positive and negative controls in each assay, respectively. Five-microliter aliquots of PCR product were analyzed by electrophoresis on 2% (w/v) agarose gel (BIOWEST, Nuaillé, France) using a PowerPac Basic Power Supply (Bio-Rad Laboratories, CA, USA). Gels were stained with GelStain (TransGen Biotech, Beijing, China) and visualized under UV light using an ImageQuant 300 Imager (GE Healthcare, Buckinghamshire, UK).
8
Embryo Protein Extraction and Western Blotting
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We dechorionated embryo collection, squashed them with a needle and pulsed with ultrasound for 30 s, added 25 ml 4× SDS sample buffer, heated for 9 min at 96°C, centrifuged at 11,000 rpm for 20 min. Lysate was stored in a fresh cup at −80/–20°C. Schneider’s cells were manually detached, centrifuged at 900 rpm, PBS was replaced by 35 µl 4× SDS sample buffer and heated 9 min at 96°C, and stored at –80°C. For supernatant samples, we centrifuged at 15,000 rpm at 4°C for 20 min, washed with acetone and resuspended in 100 µl 1× SDS sample buffer, heating it at 96°C for 9 min. We used 4–20% gradient Mini-Protean TGX Precast Protein Gels (Bio-Rad) together with PageRuler prestained protein ladder (Thermo Scientific), MiniProtean chamber (Bio-Rad), PowerPac Basic power supply (Bio-Rad) for 40 min at 170 V. The gels were then equilibrated in transfer buffer and packed into a western blotting sandwich. Sandwich blotting was performed with a PVDF transfer membrane with 0,2 µm pore size (Thermo Fisher Scientific) for embryos lysates or in other cases, Amersham Hybond-ECL membrane with 0.2 µm pore size (GE Healthcare). Protein transfer to the membrane was performed on ice in a MiniProtean chamber (Bio-Rad) that was filled with transfer buffer at 300 mA for 90 min.
9
Protein Purification Protocol Optimization
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The insoluble total protein, soluble total protein and purified MGSs from each strain were prepared as samples during the protein purification steps. All samples were diluted to a final concentration of 10 µg protein per 16 µl and mixed with 4 µl of 5 × SDS-PAGE sample buffer (Biosesang, Korea). After boiling (5 min, 95 °C), the samples were cooled on ice. 20 µl of the samples or 7 µl of ProSieve Color Protein Marker (Lonza, Swiss) were loaded to each lane of 4–12% Tris gels (Bio-Rad, USA). Gel electrophoresis was carried out in Tris–glycine-SDS buffer (0.3% Tris, 1.4% glycine, 0.1% SDS (w/v)) at 200 V using PowerPac Basic Power Supply (Bio-Rad, USA). Coomassie Brilliant Blue G-250 Staining Solution (3 M, USA) was used to stain the gels with microwaves, followed by destaining for 1–2 h in 30% methanol / 10% acetic acid (v/v) destaining buffer with shaking (50 rpm).
10
Anti-HER2 mAb Purification and Characterization
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Small-scale anti-HER2 mAb was purified using NAb Protein A Plus Spin Kit. Large-scale mAb purification using Bio-Rad NGC system (Bio-Rad, Hercules, CA) equipped with a UNOsphere SUPrA column was conducted according to the manufacturer’s protocols, including column equilibration, sample loading, column washing, and antibody elution. The equilibration buffer was comprised of 0.02 M sodium phosphate and 0.02 M sodium citrate at pH 7.5. Elution buffer contained 0.02 M sodium citrate and 0.1 M sodium chloride at pH 3.0. The pH of eluted mAb was neutralized to 7.0 with 1 M Tris solution. The mAb purity was examined by SDS-PAGE under natural condition using NuPAGE 4–12% Bis-Tris Protein Gels (1.0 mm, 10-well) and a PowerPac Basic Power Supply (Bio-Rad). The surface binding of our anti-HER2 mAb was analyzed using BD LSRII flow cytometer (BD Biosciences, San Jose, CA) after incubating cells with 1 μg of Alexa Fluor 488-labeled mAb/million cells on ice for 30 min.
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