Amersham ecl prime blocking reagent

Ovaries were collected from 40-day-old CD1 mice, frozen and stored at −20 °C. Total protein lysate was extracted with a pestle and motor using cell lysis buffer (Cell Signaling Technology, 9803S) in the presence of a protease inhibitor cocktail (Life Technologies, 78440). Denaturing SDS–PAGE was performed with 25 μg of the total protein lysate using standard western blotting techniques on a 4–15% polyacrylamide gel (Bio-Rad, 456–1084). The polyvinylidene fluoride membrane was blocked following transfer using 3% Amersham ECL Prime Blocking Reagent (GE Healthcare, RPN418) and incubated in primary antibodies as follows: 1:1,000 HMGB1 (Abcam, ab18256) and 1:1,000 GAPDH (Cell Signaling Technologies, 5174S) in TBS+0.1% Tween-20. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Membranes were then incubated in horseradish peroxidase conjugated anti-rabbit secondary antibody (GE Healthcare, NA931-1ML), and protein was visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232).

Proteins from the culture supernatant were precipitated as described^{78 (link)}. After air drying, the precipitate was resuspended in 50 mM HEPES, 2% SDS (pH 7.5) and disulfide bonds within proteins were reduced with 25 mM dithiothreitol (DTT), followed by alkylation of the thiol groups with 60 mM iodoacetamide. For Western blot analyses, 7.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE Ready Gel^® Tris-HCl Precast Gels, BioRad, CA, USA) was run at 100 V for 1 h 30 min and blotted to polyvinylidene fluoride (PVDF) membranes (Amersham Hybond P 0.45 µm pore size PVDF blotting membrane, GE Healthcare) in a Trans-Blot^® SD Semi-Dry Transfer Cell (BioRad) for 2 h with 1.8 mA/cm^{2 (link)} membrane. The membrane was blocked for 1 h at room temperature in TBS containing 4% Amersham ECL Prime Blocking Reagent (GE Healthcare) and 0.1% Tween 20. It was then incubated with polyclonal anti-FIX antibody (F0652, SIGMA) (1:3000) overnight at 4 °C. Afterwards, the membrane was washed three times with TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-linked anti-rabbit IgG (NA934, GE Healthcare) at a dilution of 1:10,000 for 1 h. The blot was washed again and proteins were detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer’s instructions.