Primary NHBE cells were lysed on ice with a cell scraper and radio-immunoprecipitation assay lysis buffer (Pierce) supplemented with protease inhibitor tablets (Roche) and a phosphatase inhibitor cocktail (Pierce). Extracts were incubated for 45 minutes for complete lysis and centrifuged at 10,000 RPM for 10 minutes at 4°C to pellet cell debris. Supernatant protein concentrations were quantified using a BCA Protein Assay kit (Pierce) and a spectrophotometric plate reader (BioTek). Most protein lysates were separated by running 30–100 μg of lysate on 10% Bis-Tris NuPage gels (Invitrogen) and subsequently transferred to 0.2 μm pore polyvinylidene fluoride (PVDF) membranes (BioRad). Primary antibody (Table S2 ) incubations were according to manufacturer’s recommendations in 0.1% Tween TBS supplemented with 5% non-fat dry milk or BSA, as recommended. Immunoreactive bands were visualized using the appropriate horseradish peroxidase-conjugated anti-IgG antibodies (Pierce). Bands were detected using enhanced chemiluminescence or prime detection reagent (GE Healthcare) whenever appropriate.
0.2 μm pore polyvinylidene fluoride pvdf membranes
Manufactured by Bio-Rad
The 0.2 μm pore polyvinylidene fluoride (PVDF) membranes are a type of lab equipment used for filtration and separation processes. These membranes have a pore size of 0.2 micrometers and are made of polyvinylidene fluoride, a durable and chemically resistant material. The primary function of these membranes is to provide a physical barrier for the separation and purification of various substances, such as proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
2 protocols using 0.2 μm pore polyvinylidene fluoride pvdf membranes
1
Protein Extraction and Immunoblotting Protocol
2
Protein Extraction and Immunoblotting Protocol
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Primary NHBE cells were lysed on ice with a cell scraper and radio-immunoprecipitation assay lysis buffer (Pierce) supplemented with protease inhibitor tablets (Roche) and a phosphatase inhibitor cocktail (Pierce). Extracts were incubated for 45 minutes for complete lysis and centrifuged at 10,000 RPM for 10 minutes at 4°C to pellet cell debris. Supernatant protein concentrations were quantified using a BCA Protein Assay kit (Pierce) and a spectrophotometric plate reader (BioTek). Most protein lysates were separated by running 30–100 μg of lysate on 10% Bis-Tris NuPage gels (Invitrogen) and subsequently transferred to 0.2 μm pore polyvinylidene fluoride (PVDF) membranes (BioRad). Primary antibody (Table S2 ) incubations were according to manufacturer’s recommendations in 0.1% Tween TBS supplemented with 5% non-fat dry milk or BSA, as recommended. Immunoreactive bands were visualized using the appropriate horseradish peroxidase-conjugated anti-IgG antibodies (Pierce). Bands were detected using enhanced chemiluminescence or prime detection reagent (GE Healthcare) whenever appropriate.
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