Goat anti mouse hrp antibodies

Eight-week-old female NZM and FVB mice were administered three subcutaneous injections of tamoxifen-induced apoptotic bodies or cisplatin-induced apoptotic bodies (20 μg in Incomplete Fruend’s Adjuvant (IFA)), at fortnightly intervals. Control animals received IFA. Sera (at 5 weeks) were assessed for the presence of autoantibodies by ELISA and Western blot. For ELISA, autoantigens (Ro60, RNP A, RNP 68, dsDNA; Arotec Diagnostics Limited) were individually adsorbed onto ELISA plates (1 μg/well) and incubated with pooled sera (diluted 1:1000 in PBS containing 1% BSA and 0.1% Tween-20) for 16 hrs at 4°C. Following further incubation with appropriately diluted goat anti-mouse-HRP antibodies (Jackson ImmunoResearch), enzyme activity was visualized by addition of 3,3′,5,5′-Tetramethylbenzidine (TMB; Invitrogen). For Western blot, nitrocellulose strips (containing moieties in LLC1 lysate, resolved on SDS-PAGE) were incubated with pooled sera, diluted 1:1000 in PBS containing 1% BSA and 0.1% Tween-20. After further incubation with an appropriately-diluted goat anti-mouse HRP conjugate (Jackson ImmunoResearch), enzyme activity was visualized by enhanced chemiluminescence (ECL; Pierce). Antibodies to β-actin (Santa Cruz Biotechnology) were employed to verify equivalence of protein content.

For detection of gPrestin expressed in Sf9 cells, samples were separated on 4–20% gradient Mini-Protein precast gels (BioRad), transferred to 0.45 μm nitrocellulose membranes (BioRad) and probed with 1:5000 dilutions of anti-V5 antibodies (Invitrogen) followed by 1:5000 dilutions of goat anti-mouse-HRP antibodies (Jackson Laboratory). For detection of gPrestin expressed in HEK293T cells, anti-GFP (Rockland) and anti-chicken-HRP (Jackson Laboratory) antibodies were used.