Before the electrophoresis was started, 20 µL of Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) was added to the crude and partially purified samples. The samples were then heated by using a digital dry bath to 98 °C and were kept at that temperature for 5 min. For the electrophoresis, Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA, USA), Mini PROTEAN Tetra Cell (Bio-Rad, Hercules, CA, USA), and the PowerPac HC (Bio-Rad, Hercules, CA, USA) power supply were used. The buffer tank was filled with the reused 1× running buffer, while the reservoir between the gels was filled with a freshly prepared and 10 times diluted 10× running buffer (30.3 g L−1 TRIZMA base, 144 g L−1 glycine, 10 g L−1 SDS (Sigma Aldrich, St. Louis, MO, USA)). One well was used for 15 µL of the unstained Precision Plus Protein Standard (Bio-Rad, Hercules, CA, USA) while the other four wells were loaded with 15 µL of the denatured samples. The samples were run at 120 V for about an hour. After the electrophoresis was finished, the gels were stained using QC Colloidal Coomassie (Bio-Rad, Hercules, CA, USA) and washed using distilled water. A picture of the gel was taken using the Universal Hood II Gel Doc System (Bio-Rad, Hercules, CA, USA).
Powerpac hc
Manufactured by Bio-Rad
Sourced in United States
The PowerPac HC is a high-current power supply designed for a variety of electrophoresis applications. It provides constant voltage, constant current, and constant power modes to support various gel electrophoresis techniques. The device features a wide range of voltage and current outputs to accommodate different sample sizes and separation requirements.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tetra cell, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Gel doc ez imager, by Bio-Rad (2 mentions)
Anti ki67 antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti histone h3 antibody, by Cell Signaling Technology (2 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Cell Signaling Technology (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
Qc colloidal coomassie, by Bio-Rad (1 mentions)
Universal hood 2 gel doc system, by Bio-Rad (1 mentions)
Nupage tris acetate sds running buffer, by Thermo Fisher Scientific (1 mentions)
Bio safe cbb, by Bio-Rad (1 mentions)
Wide view prestained protein size marker 3, by Fujifilm (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Quick load purple 2 log dna ladder, by New England Biolabs (1 mentions)
Typhoon 9210, by GE Healthcare (1 mentions)
26 protocols using powerpac hc
1
SDS-PAGE Protein Separation and Visualization
2
SDS-PAGE Protein Separation
3
Agarose Gel Electrophoresis of PCR Products
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We analyzed 10μL of each PCR product using 1% agarose electrophoresis in TAE (Tris-Acetic Acid-EDTA) buffer (Sigma-Aldrich, MO, USA). Gels were dyed with GelGreen (CA, USA) using a 1:10,000 dilution and a current of 110 V supplied by a Bio-Rad PowerPac HC power supply (Bio-Rad, CA, USA) for 50 minutes. We used the Quick-Load Purple 2-Log DNA Ladder (NEB, MA, USA) as molecular a molecular weight marker. We analyzed the gels by UV transillumination using a Bio-Rad ChemiDoc XRS imaging system.
In addition, in some of our experiments, we used the blueGel unit, a portable electrophoresis unit sold by MiniPCR from Amplyus (MA, USA). This is a compact electrophoresis unit (23 × 10 × 7 cm) that weighs 350 g. In these experiments, we analyzed 10μL PCR product using 1% agarose electrophoresis tris-borate-EDTA buffer (TBE). Gels were dyed with GelGreen (CA, USA) using a 1:10,000 dilution, and a current of 48V was supplied by the bluegel built-in power supply (AC 100-240V, 50-60hz).
In addition, in some of our experiments, we used the blueGel unit, a portable electrophoresis unit sold by MiniPCR from Amplyus (MA, USA). This is a compact electrophoresis unit (23 × 10 × 7 cm) that weighs 350 g. In these experiments, we analyzed 10μL PCR product using 1% agarose electrophoresis tris-borate-EDTA buffer (TBE). Gels were dyed with GelGreen (CA, USA) using a 1:10,000 dilution, and a current of 48V was supplied by the bluegel built-in power supply (AC 100-240V, 50-60hz).
4
Quantifying protein S-nitrosation via fluorescence
5
Detailed Spectroscopic Analysis Protocol
6
Western Blot Analysis of gD Expression
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Electrophoresis was performed with 10% polyacrylamide sodium dodecyl sulfate (SDS) gels, for 60 minutes at 20 V and 100 mA (PowerPac HC; Bio-Rad Laboratories). Gels were loaded with 20 μL of transfected cell lysate in the loading dye. After running, the gels were transferred to a methylcellulose membrane for 45 minutes at 20 V and 400 mA on a Bio-Rad blotter and labeled with an anti-gD mAb or an anti-BoHV-1 polyclonal Ab. Briefly, the membranes were blocked with PBS with Tween-20 (PBST) plus 3% low fat dehydrated milk (blocking buffer), overnight at 4°C with constant rocking. Membranes were incubated with mouse anti-gD mAb (VIDO) in blocking buffer at an appropriate dilution, for 60 minutes at RT with constant rocking. Membranes were washed three times with PBST and incubated in the same conditions with biotinylated goat anti-mouse IgG (H+L), human serum-adsorbed polyclonal Ab (Kirkegaard and Perry Laboratories, Inc, Gaithersburg, MD, USA). Membranes were washed again in the same way and incubated as previously described with streptavidin-alkaline phosphatase. Membranes were washed five times with PBST and developed with nitro-blue-tetrazolium-5-bromo-4-chloro-3-indolyl-phosphate (NBT-BCIP) (Promega). ImageJ freeware image processing developed at the National Institutes of Health was used for image analysis (http://rsbweb.nih.gov/ij/ ).
7
Liver Tissue Protein Extraction and Western Blot Analysis
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The liver tissue (0.1 g) was cut with scissors, ground in RIPA buffer (0.9 mL) with an electric homogenizer, and placed on ice for 2 h. After centrifuging the homogenate at 12,000×g and 4 °C for 15 min, the protein concentration was measured using the BCA method. RIPA buffer was then added to balance the protein concentration in the liver tissue homogenate. All samples were mixed with 2 × loading buffer, boiled for 10 min, and separated using SDS–PAGE at 50 V for 30 min, followed by 100 V for 90 min using an electrophoresis instrument (PowerPac HC, Bio-Rad Laboratories). Proteins in the gel were transferred the protein to a polyvinylidene fluoride (PVDF) membrane for 20–40 min. The PVDF membrane was blocked with 5% skimmed milk at 25 °C ± 1 °C for 2 h; antibodies against NF-κB (1:1000), HSP70 (1:1000), caspase-3 (1:1000), and β-actin (1:1000) were added and incubated overnight at 4 °C. The membranes were washed five times with TBST (5 min each) and incubated with the corresponding second antibody (1:20,000) to 25 °C ± 1 °C. After washing the PVDF membrane three times with TBST solution, bands were visualized via chemiluminescence (ChemDoc-IT®510 Imager; Ultra-Violet Products Ltd., Cambridge, UK). The membranes were scanned, and band intensities were analyzed using Visionworks LS (version 8.1.2; Ultra-Violet Products Ltd.).
8
Rapid Tissue Clearing and Staining
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Rapid clearing of fixed biopsy tissues was achieved with a modified SmartClear II pro (Life Canvas Technologies) with an external 300 W electrophoresis PowerPac HC power supply from BioRad to apply higher voltage (250V). In order to maintain the temperature below 70°C, all insulation material in SmartClear II pro was removed and the device was operated in a cold room at 4°C. Operational parameters for the SmartClear device were 4~6 V/mm for electric field strength, 0.1 rpm for electric field rotation frequency, and 70°C for clearing temperature. 3% (w/v) SDS buffer titrated to 9.5 pH was used as the clearing buffer. The cleared biopsy tissues were stained using a custom device (Kim, PNAS, 2015). Operational parameters were 8~10 V/mm for electric field strength, 0.1 rpm for electric field rotation frequency, and 30°C for staining temperature. 20 mM Tris buffer titrated to pH 7.5 with 20% (w/v) D-sorbitol and 1% (w/v) BSA was used as the staining buffer. The samples were stained for 1 hr with 5 μL of 10X concentrated lectin-594(DL-1177, Vector laboratories, CA) and 15 μL of rabbit anti-histone-H3 antibody (12230S, Cell Signaling Technology, MA) in 4 mL of staining buffer. 20 μL of anti-Ki-67 antibody (11882S, Cell Signaling Technology, MA) was used for labeling mouse kidney biopsy tissue.
9
Serum Protein Electrophoresis Using SDS-PAGE
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The regular glycine SDS-PAGE and tricine SDS-PAGE preparation were described as in standard protocols [28 (link),29 (link)].
For electrophoresis analysis, the 1 μL of serum sample containing 65 μg proteins was diluted 1:2 ratio with 2 μL of denaturing solution (8 M urea, 10 mM IAA). The sample was then incubated at room temperature for 30 min before centrifugation at 14,000 × g for 5 min. The supernatant was diluted 4:1 ratio with SDS-PAGE loading buffer (1 M Tris-HCl, pH6.8, 10% SDS, 50% glycerol, and 1% bromphenol blue). After mixing and centrifugation at 13,800 × g for 5 min, the supernatant was transferred to a new tube and was ready for gel electrophoresis.
For regular glycine SDS-PAGE analysis, electrophoresis was carried out at a constant 80 V for 3 hours (PowerPac HC, Bio-Rad). Then the gel was briefly rinsed with ultrapure water and stained with Coomassie brilliant blue G-250. After destaining, gels were scanned with a Scanjet image system (HP Scanjet G4050). Gel images were analyzed using Scion Image (http://rsb.info.nih.gov/nihimage/ ).
For tricine SDS-PAGE analysis, the prepared samples were separated at a constant 30 V until they completely entered into the separating gel from the stacking gel. Then a constant 200 V was maintained until the tracking dye reached the bottom of the gel. The staining, destaining, and image analysis were performed as described above.
For electrophoresis analysis, the 1 μL of serum sample containing 65 μg proteins was diluted 1:2 ratio with 2 μL of denaturing solution (8 M urea, 10 mM IAA). The sample was then incubated at room temperature for 30 min before centrifugation at 14,000 × g for 5 min. The supernatant was diluted 4:1 ratio with SDS-PAGE loading buffer (1 M Tris-HCl, pH6.8, 10% SDS, 50% glycerol, and 1% bromphenol blue). After mixing and centrifugation at 13,800 × g for 5 min, the supernatant was transferred to a new tube and was ready for gel electrophoresis.
For regular glycine SDS-PAGE analysis, electrophoresis was carried out at a constant 80 V for 3 hours (PowerPac HC, Bio-Rad). Then the gel was briefly rinsed with ultrapure water and stained with Coomassie brilliant blue G-250. After destaining, gels were scanned with a Scanjet image system (HP Scanjet G4050). Gel images were analyzed using Scion Image (
For tricine SDS-PAGE analysis, the prepared samples were separated at a constant 30 V until they completely entered into the separating gel from the stacking gel. Then a constant 200 V was maintained until the tracking dye reached the bottom of the gel. The staining, destaining, and image analysis were performed as described above.
10
Rapid Tissue Clearing and Staining
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