Secondary immunoglobulin g igg conjugated with horseradish peroxidase

Western blot analysis was carried out for different HCN proteins (1-4) in the bladder tissue. Briefly, bladder tissue was homogenized using CelLytic™ MT Mammalian Tissue Lysis/Extraction Reagent (Sigma, USA) in the presence of phenylmethylsulfonyl fluoride (1 mM), sodium orthovanadate (2 mM) and protein inhibitor cocktail (Sigma, USA). Protein estimation was done by BCA Protein Assay Kit (Pierce, Rockford, Illinois). An equal amount (50 μg/well) of denatured proteins was loaded in 10% tricine-SDS gel and blotted on polyvinylidene fluoride (PVDF) membranes using wet transfer system. After blocking (2 h at 37°C), membranes were incubated overnight at 4°C with primary antibodies specific for HCN1, HCN4 and Actin-β (Santa Cruz Biotechnology), HCN2 and HCN3 (Abcam, USA), in blocking buffer (pH 7.5). The membranes were then re-incubated for 2 h at room temperature with secondary immunoglobulin G (IgG)-conjugated with horseradish peroxidase (Santa Cruz Biotechnologies, USA). The blots were developed using luminol (Thermo Scientific, USA) and measured on Versa doc imaging system (Model 4000; BioRed, USA). Densitometry for protein specific band was done using AlphaEase FC StandAlone V. 4.0.0 software. β-Actin was used as an internal control to normalize the band density.