Amersham hybond ecl

The proteins were denatured by boiling for 5 minutes with loading buffer (125 mM Tris-HCl, 12% (w/v) SDS, 10% (v/v) glycerol, 22% (v/v) β-mercaptoethanol, and 0.001% (w/v) bromophenol blue) and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were either visualized by Coomassie blue staining or electrophoretically transferred to polyvinlidene difluoride (PVDF) membrane (Amersham Hybond-ECL; Amersham Biosciences, UK). The membrane was blocked with 5% non-fat dried milk, 0.1% Tween20 in PBS (PBST). The membrane was probed with mouse monoclonal anti-OPN antiserum (Abcam, UK) diluted 1:5,000 in 1% non-fat dried milk in PBST and goat anti-mouse IgG-HRP conjugated (Sigma, USA) diluted 1:10,000 in 1% non-fat dried milk in PBST. The membranes were developed by chemiluminescence using ECL plus detection reagent (GE Healthcare, UK).

The cells were lysed 72 h after transfection in RIPA buffer, as well as in protease and phosphatase inhibitors (Sigma Aldrich Gmbh, Steinheim, Germany). Bradford assay determined the total protein concentration. Total protein from the lysates (30 μg) was resolved by 12% SDS-PAGE and, after electrophoresis, gels were blotted onto nitrocellulose membranes. Nonspecific binding sites were blocked for 2 h with 0.1% bovine serum albumin or 5% nonfat dried milk in TBST (TRIS-buffered saline solution containing 1% Tween 20). All washes and antibody incubations were performed using TBST. The following primary antibodies were used: anti-Cyclin E1 (Sta Cruz sc-481) 1 : 500 in blocking buffer (0.1% bovine serum albumin in TBST), anti-SHP (Perseus Proteomics-PP-N7519-00) 1 : 1000 in blocking buffer (5% nonfat dried milk in TBST), and anti-actinin 1 : 1000 in TRIS-buffered saline containing 1% Tween 20. Proteins were visualized by ECL detection with secondary HRP-conjugated anti-rabbit (Amersham Hybond ECL, Freiburg, Germany) or anti-mouse antibodies (Jackson Immuno Research, Pennsylvania, USA). Immunoblot results were quantified by densitometer using the GeneSnap and GeneTools software (SynGene-Synoptic Ltd., Cambridge, UK). Ponceau staining of membranes was used to monitor protein transfer and loading.