Hybond ecl extra

The electrophoretic experiments were performed by SDS-PAGE (10 % polyacrylamide unless specified otherwise) to analyze the YKL-40 and EMT related genes. Electrophoresis was conducted by a vertical gel electrophoresis device that was powered by (Mini PIII, Bio-Rad, Hercules, CA) a PAC 300 power supply (Bio-Rad). All SDS-PAGE samples (20 μg) were equilibrated in 10 mM Tris–HCl and 5 % SDS (pH 7.6) before loading.
Following complete separation, the gel was soaked briefly in a transfer buffer, which contained 25 mM Tris, 192 mM glycine, 20 % methanol, and 0.0375 % SDS (pH 8.3), for 30 s. The gel was then immediately electrotransfered to a nitrocellulose membrane (Hybond-ECL extra; Amersham) at 90 mA for 60 mins in a semi-dry Transfercell (Bio-Rad). The membrane was immersed in 2 % skim milk for 1 h with gentle agitation. After three washes with PBS for 5 mins each, the membrane was subjected to react with monoclonal or polyclonal antibodies and developed with chemiluminescence agents.

The gel from the SDS-PAGE was soaked instantly and briefly in a transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol (v/v), and 0.0375% SDS (pH 8.3)) for 30 s [4 (link), 36 (link)]. Then, the gel was immediately electrotransferred to a nitrocellulose membrane (Hybond-ECL extra, Amersham, Buckingham, UK) at 90 mA for 1 hour using a semidry transfer cell (BioRad, Hercules, CA). The membrane was immersed in 1% gelatin for 1 hour with gentle shaking. After 3 washes with PBS for 5 min each, the membrane was incubated with 1 mg of LG in a buffer (PBS with 0.5% gelatin and 0.05% Tween-20) for 1 hour. After 3 washes, the blot was incubated with mouse anti-LG conjugated to horseradish peroxidase (anti-LG-HRP) for 1 hour. Finally, the membrane was developed with 0.1 mg/mL of 3-3′-diaminobenzidine (3,3′,4,4′-tetra-amino-biphenyl) containing 0.01% H₂O₂ in PBS.