Anti goat hrp conjugated antibody

Purified His-tagged proteins (4 μg) were subjected to SDS-PAGE and then blotted onto Immobilon-P membrane (Millipore). The membranes were blocked in 10% low fat milk in PBS with 0.05% Tween 20 to avoid non-specific interactions and then incubated either with purified GST fused recombinant proteins or GST alone (4 μg). After five washings the membranes were incubated with an antibody against GST (anti-GST goat polyclonal, GE Healthcare, 1∶1000 dilution) and anti-goat HRP conjugated antibody (Santa Cruz Biotechnology Inc., 1∶5000 dilution). The signals were developed using ECL (GE Healthcare).

Cells were lysed and protein extracts boiled and loaded on 8% polyacrylamide gels. After electrophoresis, proteins were transferred to PVDF membranes, which were blocked for 1 h in 5% milk powder before primary antibody was applied at 4°C overnight. The membranes were washed and stained with secondary antibody. Enhanced chemo-luminescence was elicited using ECL Western Blotting Substrate (Thermo Scientific, Schwerte, Germany) according to the manufacturer's instructions. The following primary antibodies were used: anti-CASPASE3, anti-CASPASE8, anti-CASPASE9, anti-PARP (all Cell Signaling Technology, Boston, MA, USA); anti-POLD1 (sc-8797, Santa Cruz Biotechnology, Heidelberg, Germany). Anti-β-ACTIN antibody (Sigma-Aldrich) served as loading control. The following secondary antibodies were used: anti-goat HRP-conjugated antibody (Santa Cruz Biotechnology); anti-mouse and anti-rat HRP-conjugated antibody (GE Healthcare, Freiburg, Germany).