Canoscan 9900f scanner

For quantification of protein levels after miRNA expression, transfected MiaPaCa cells were fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Braunschweig, Germany). 20 μg of protein from the nuclear fraction was loaded onto a 10% polyacrylamide gel and was then electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with Tween-20 (0.05%)-TBS (pH 7.4; 0.1 M Tris Base, 1.4 M NaCl) containing 5% milk, followed by incubation with respective primary antibody α-TCF4 (LS-Bio, Eching, Germany) at a concentration of 1:1000 or as a control α-β-Actin (Abcam, Cambridge, UK) with a concentration of 1:2000. Membranes were washed with Tween-20 (0.05%)-TBS and were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:5000). Signals were detected using the enhanced chemiluminescence system (ECL, Amersham Life Science Ltd., Bucks, UK). Films were scanned with a CanoScan 9900F scanner (Canon, Japan). Protein levels were quantified using the Odyssey software LI-COR and normalized to the β-Actin control.

In brief, 20–80 μg of total cell lysate was loaded onto a 10% polyacrylamide gel and was then electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with 20 ml of Tween-20 (0.05%)-TBS (pH 7.4; 0.1 M Tris Base, 1.4 M NaCl) containing 3% or 5% milk for 1 h, followed by incubation with respective primary antibody overnight at 4°C. Membranes were washed 3 times with Tween-20 (0.05%)-TBS and were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit anti body (1:3000) for 1 h at room temperature. Signals were detected using the enhanced chemiluminescence system (ECL, Amersham Life Science Ltd., Bucks, UK). Films were scanned with a CanoScan 9900F scanner (Canon, Japan). Densitometric analysis was performed using the ImageJ software (http://imagej.nih.gov/ij).