Horseradish peroxide hrp conjugated anti mouse igg

PDAC cells (1×10⁷) from the various cell lines were harvested, lysed, resolved and transferred to nitrocellulose membranes, as previously described [40 (link)]. Membranes were incubated for 1 h at RT with anti-ENO1 72/1 mAb or rabbit polyclonal anti- β-Actin antibody (Sigma-Aldrich), at dilutions of 1:2000 in Tween-Tris-Buffered Saline (TTBS) and then probed with a horseradish peroxide (HRP)-conjugated anti-mouse IgG (Santa Cruz) or HRP-conjugated goat anti-rabbit Ig secondary antibody (Sigma-Aldrich) at dilutions of 1:2000. For Western blot analysis of ENO1 in PANC-1/P, PANC-1/M and HPDE cells, membranes were probed with in-house purified rabbit antiserum against ENO1 or with mouse antibody specific to β-Actin (Sigma St. Louis, MO, USA) as a protein loading control. Immunocomplexes were detected by probing with appropriate secondary antibodies conjugated with HRP (Jackson ImmunoResearch), and were visualized using the SuperSignal detection system (Thermo Fisher, Waltham, MA, USA).

Lysates were prepared using the Active Motif kit protocol. Proteins were quantified by Bradford assay (Bio-Rad) according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The gels were blotted onto a Nitrocellulose Membrane (Bio-Rad) and blocked with 5% non-fat dry milk and reacted with the appropriate antibodies for BAF subunits: anti-BAF57/SMARCE1 antibody (Abcam ab131328) and anti-SNF5/SMARCB1 antibody (Abcam ab126734), which are rabbit monoclonal against the human subunits and are known to cross-react with zebrafish Smarce1 and Smarcb1 respectively, all membranes were incubated with anti-ERK2 antibody (Santa Cruz Biotechnology, USA; Cat. Nr. sc-153) to confirm equal protein loading. The blots were posteriorly incubated with horseradish peroxide HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology). Chemiluminescence was detected with enhanced chemiluminescence (ECL) western blot detection kits (Thermo Scientific Pierce).