Trueblot anti rabbit igg hrp

Culture medium or serum samples were diluted to a final volume of 500 μL with ice-cold phosphate-buffered saline containing 0.2% Nonidet P-40 (PBS-NP40) and incubated with 1 μL of rabbit anti-LCAT monoclonal antibodies (Abcam) for 18 h at 4 °C with gentle rotation. After the first immunoreaction, 10 μL of TrueBlot anti-Rabbit Ig IP Beads were added and incubated with rotation for 2 h at 4 °C. The IP Beads were pelleted by centrifugation, washed with PBS-NP40, and the immunoprecipitates were eluted by boiling in 10 μL of Laemmli sample buffer. The immunoprecipitates were electrophoresed and subjected to immunoblotting. Purified human LCAT (Roar Biomedical Inc.) or human plasma HDL (Merck Calbiochem) was used as a positive control. Rabbit anti-LCAT polyclonal antibodies (Novus Biologicals) and TrueBlot anti-Rabbit IgG HRP (1:5000) (eBioscience) were used as primary and secondary antibodies, respectively. HRP reactions were performed by incubation with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher), and chemiluminescence was detected using a ChemiDoc imaging system (Bio-rad).

Native-PAGE analysis of serum hemoglobin/haptoglobin complex was performed using Novex Wedgewell 4%–20% Tris-Glycine gels (1.0 mm × 15 wells, Thermo Fisher). Serum samples were mixed with an equal volume of 2X Native Tris-Glycine Sample Buffer (Invitrogen) and electrophoresed using Tris-Borate-EDTA buffer for 3 h with a constant voltage of 100 V at 4 °C. The gels were stained with Coomassie Brilliant Blue G250 or subjected to immunoblotting using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) for protein transfer. Human hemoglobin and haptoglobin were detected with anti-hemoglobin (1 μL/5 mL TBST) and anti-haptoglobin (1 μL/5mL TBST) antibodies, respectively. TrueBlot anti-rabbit IgG HRP (1:5000) (eBioscience) or TrueBlot anti-mouse IgG HRP (1:5000) (eBioscience) were used as secondary antibodies. HRP reactions were performed by incubation with Immobilon Forte Western HRP substrate (Merck Millipore), and chemiluminescence was detected using a ChemiDoc imaging system (Bio-rad).