alpha-Smooth Muscle Actin Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Goat (1:100) --Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Protocol tips
-Goat (1:100) --Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

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).

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Manufacturer protocol

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