Ab765p

Frozen liver tissue was homogenized in 50 mM HEPES, 150 mM NaCl, 10% Glycerol, 0.1% Tween 20, 7.5 mM EDTA, and 7.5 mM MgCl₂*6H₂O. Protein content was determined using a DC^™ Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA), and homogenates were diluted to 5 mg/ml. To reduce nonspecific binding of the anti-collagen type I antibody, liver homogenates were digested with pepsin prior to electrophoresis. To accomplish this, pepsin (Powder 1:3000; VWR, Radnor, PA) was diluted to 2 mg/ml in 2 N HCl, and 10 μl of this pepsin preparation was added to 100 μl of liver homogenate (500 μg protein). This sample was incubated for 2 hours at 20°C and then neutralized with 10 μl of 2 N NaOH and resuspended in SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 0.2% bromophenol blue and 20% glycerol) containing 400 mM β-mercaptoethanol. pepsin-digested samples (6 μl/lane) were resolved on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and incubated with an anti-collagen type I antibody (#AB765P, EMD Millipore, Hayward, CA). Undigested liver homogenates (25 μg protein/lane) were probed with an anti-actin antibody (Santa Cruz Biotech, Dallas, TX) and served as a quasi-loading control. Blots were subsequently incubated with species-specific, HRP-conjugated secondary antibodies, and bands were visualized with Pierce^™ ECL Western Blotting Substrate (Thermo Scientific).