Chemidoc xrs detection system ecl

The concentrations of total protein extracted with the RIPA reagent were detected by the BCA protein assay kit. Protein extracts were diluted, boiled with SDS PAGE loading buffer at 95 °C for 10 min and then were subjected to protein separation by loading onto 10% SDS PAGE. Proteins in the gel were transferred to a PVDF membrane. The membranes were blocked with 5% skimmed milk powder in tris buffered saline with 0.05% Tween 20 (TBS-T) for 2 h at room temperature. After a short wash with TBS-T, the membranes were incubated with specific primary antibodies which were diluted in TBS-T by 200 to 5,000 folds overnight at 4 °C. The membranes were washed four times with TBS-T and then were incubated with secondary antibody diluted in TBS-T by 2500- or 5000-fold for 1 h at room temperature. After washing four times with TBST, the detection of proteins was performed using the ChemiDoc XRS + detection system (ECL, Bio-Rad). The immunoblots were analyzed with the Quantity One^® Image Analyzer software program (Bio-Rad). β-actin was used as an internal reference for examined proteins except membrane UT-A1. Equal loading of protein amounts while evaluating membrane UT-A1 was demonstrated using Ponceau staining.