Tissues were homogenized in RIPA buffer (final concentration: 50 mM Tris, 150 mM NaCl, 1% Triton-X100, 0.1% SDS, 0.5% sodium deoxycholate, with Complete protease inhibitors (Roche)) and samples were incubated for 1 h rotating at 4 °C then clarified by centrifugation at 10,000g for 10 min. Total protein concentration was determined by DC protein assay (BioRad) and 30 μg was loaded onto 4–12% NuPAGE Bis-tris gels in MES buffer (Novex Life Technologies) to determine eGFP and β-catenin levels. After electrophoresis, proteins were transferred to 0.2 μm PVDF (Bio-Rad). Membranes were blocked with 5% milk in PBS-T and then blotted with rabbit anti-β-catenin (Ab2365, dilution 1:4,000; Abcam), rabbit anti-GFP (A11122, dilution 1:5,000; Invitrogen) followed by horseradish-peroxidase-coupled antibodies (goat anti-rabbit: 111–035-144, dilution 1:50,000; Jackson ImmunoResearch). Blots were developed with ECL Plus reagents (Amersham Pharmacia) and imaged on the ChemiDoc Imaging System (BioRad). eGFP and β-catenin protein levels were determined using the same PVDF membrane (whole blot). Restore western blot stripping buffer (21063, Thermo Fisher Scientific) was used to remove primary eGFP and secondary antibodies before β-catenin detection.
0.2 μm pvdf
Manufactured by Bio-Rad
Sourced in United States
The 0.2 μm PVDF is a type of porous membrane filter made of polyvinylidene fluoride (PVDF) material. It has a nominal pore size of 0.2 micrometers, which is suitable for the filtration of small particles and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Ab2365, by Abcam (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Ecl plus reagent, by Cytiva (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Fujifilm (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (1 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
3 protocols using 0.2 μm pvdf
1
Quantifying eGFP and β-Catenin Levels
2
SDS-PAGE and Western Blot Analysis of HPV58-E7
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Cell lysates or protein samples prepared in reducing sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 0.01% Bromophenol Blue, 10% glycerol, 50 mM DTT) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (0.2 μm PVDF, Bio-Rad, U.S.A.) membranes. After blocking with 10% nonfat milk (BD DifcoTM, U.S.A.) for 1 hour, the membranes were incubated with antibodies specific for HPV58-E7 (1:10,000). Secondary peroxidase-conjugated goat anti-rabbit IgG (1:5000; Beyotime) was incubated for 2 hours at room temperature and ECL substrates (Millipore, U.S.A.) were applied and signals were detected by a chemiluminescence imaging system (FUJIFILM, U.S.A.).
3
Murine Norovirus Infection Assay in BV2 Cells
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BV2 cells were grown and infected in 12well plates (2 e5 cells/well). Cells were lifted with trypsin/0.25% EDTA (Gibco, ThermoFisher), washed with 1X cold PBS, resuspended in 100 μL NP40 lysis buffer (Alfo Aesar, Havervill, MA) with protease inhibitor cocktail (Sigma, St Louis, MO), incubated on ice (15min with intermittent mixing), cleared via centrifugation (1min, 4C, full-speed), and diluted 1:1 with 2x SDS loading buffer (0.125 M Tris [pH 6.8], 4% SDS, 20% glycerol, 0.004% bromophenol blue, 10% beta-mercaptoethanol). Lysates were run on Novex Tris-Glycine gels (ThermoFisher), transferred to 0.2 μm PVDF (BioRad, Hercules, CA), blocked with 5% nonfat dry milk, and probed with the following antibodies (Ab): caspase 3 (CST), PARP (CST), beta-Actin pAb (Invitrogen), MNV NS1/2 (a gift from Dr. Vernon Ward, U. of Otago, Australia), or MNV ProPol (a gift form Dr. Kim Green, NIH). Signal was detected using goat, anti-rabbit IgG-HRP (Jackson ImmunoResearch), developed with ECL substrate (Pierce, ThermoFisher), and imaged with an ImageQuant LAS4000 (GE Healthcare, Little Chalfont, UK).
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