Publication protocol
Primary cultures of keratocytes were treated with or without IL-1α (10 ng/mL) or TGF-β1 (2 ng/mL) for 16 hours. Cells were then moved to ice, rinsed three times with 2 mL of ice-cold PBS buffer, and extracted by adding 4 M guanidine chloride containing 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1 mM PMSF (Sigma), 10 μg/mL aprotinin, and protease inhibitor cocktail (Roche, Mannheim, Germany).19 Extracts were then dialyzed into 50 mM Tris/HCl, pH 7.5, containing 10 mM CaCl2 for 4 hours, and the precipitates (extracellular matrix proteins) were collected and dissolved in 0.1 M Tris-acetate solution (pH 6.0) containing 6 M urea and protease inhibitor. Protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific). For keratocan, a 100 μg protein aliquot was incubated with endo-β-galactosidase (0.1 U/mL; Sigma-Aldrich) in 50 mM sodium phosphate (pH 5.8) at 37°C overnight.9 After digestion, the protein was lyophilized, dissolved, and underwent Western blot analysis for keratocan. For perlecan Western blots, the dialyzed extract was digested with 1 mU/mL of heparitinase III (Cat No. H8891; Sigma-Aldrich) at 37°C for 3 hours.19 For Western blot analysis, 10 μg of cellular protein was separated on 4% to 15% SDS-PAGE gels and then transferred to PVDF membranes for immunoblotting. The membranes were blocked with 5% nonfat milk and probed with primary antibodies at 4°C overnight. The primary antibodies used were anti-perlecan (1:1,000, sc377219; Santa Cruz Biotechnology), anti-nidogen-1 (1:500, AF2570, R&D System), anti-nidogen-2 (1:1000, sc-373859; Santa Cruz Biotechnology), and keratocan (cat. no. sc33242, 1:2000; Santa Cruz Biotechnology). Western blotting for β-actin (1:5,000, A5441; Sigma-Aldrich) was used as a loading control. Western blot analysis was performed using enhanced chemiluminescence for signal detection. Western blot signal intensities were quantified by densitometry using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Western blotting Smooth muscle actin using alpha-Smooth Muscle Actin Antibody from Novus Biologicals. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.