Nupage novex 3 8 tris acetate gel electrophoresis

Keratinocytes were lysed in protein extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Nonidet P-40, 4 mM EDTA) containing proteinase inhibitors cocktail (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). Lysates were incubated for 30 min on ice and centrifuged at 15,000 × g for 30 min at 4°C. Supernatants were collected and protein concentrations were measured using the Bradford assay (BioRad, Hercules, CA). For each sample, 40 μg of total protein was resolved on NuPAGE Novex 3%–8% Tris-Acetate gel electrophoresis (Invitrogen, Carlsbad, CA) and electrotransferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). For type VII collagen analysis, blots were probed with a monospecific polyclonal anti C7 antibody (a generous gift from Dr A. Nystrom). Antibodies against vinculin (Abcam) or α-tubulin (Sigma, St. Louis, MO) were used as loading controls. Visualization was performed by incubating with HRP-conjugated anti-rabbit antibody (Amersham, Burlington, MA) and West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Keratinocytes were lysed in protein extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Nonidet P-40, and 4 mM EDTA) containing proteinase inhibitors cocktail (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). Lysates were incubated for 30 min on ice and centrifuged at 15,000 × g for 30 min at 4°C. Supernatants were collected and concentrated by ultrafiltration using Amicon Ultra columns (10 kDa; Millipore, Ireland). Protein concentrations were measured using the Bradford assay (Bio-Rad, Hercules, CA). For each sample, 40 μg total protein was resolved on NuPAGE Novex 3%–8% Tris-Acetate gel electrophoresis (Invitrogen, Carlsbad, CA) and electrotransferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). For type VII collagen analysis, blots were probed with a monospecific polyclonal anti-C7 antibody (a generous gift from Dr. A. Nystrom, University of Freiburg). An antibody against GAPDH was used as a loading control. Visualization was performed by incubating with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Amersham, Burlington, MA) and West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).