Peroxidase conjugated anti rabbit igg antibody

OPM2 cells (2×10⁶ per well of a 6-well plate) were incubated with ²¹³Bi-anti-CD38-MAb (0.74 MBq/ml). At different time points after start of incubation, i.e. at 0, 5, 9, 24, 48, 72, 96 and 120 h, cells were washed in PBS and subsequently lysed (50 mM Tris, pH 7.5; 250 mM NaCl; 0.1% Triton X-100; 1 mM EDTA; 50 mM NaF + protease inhibitors) at 4°C for 10 min. Lysates were centrifuged (13,500 rpm, 4°C, 10 min) and supernatants (containing 25 μg of protein each; BCA protein assay kit, Pierce, USA) were subjected to SDS-PAGE. Western blotting using different antibodies against clapsin (gift from Michele Pagano), cdc20 (Santa Cruz Biotechnology, Heidelberg, Germany), p27 (BD Biosciences, Heidelberg, Germany), Plk1 (Invitrogen / Life Technologies, Darmstadt, Germany), wee1, cyclin B1, p-HH3, BimEL, Bax, cleaved PARP (all from Cell Signaling Technology / New England Biolabs, Frankfurt, Germany), γH2AX, active caspase-3 (all from Millipore, Schwalbach/Ts, Germany), peroxidase-conjugated monoclonal anti-β-actin antibody (clone 8226, Abcam, Cambridge, UK) and peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Hatfield, UK) was performed as described previously [42 (link)].

1x10⁷ parasites were lysed in 1x LDS buffer (Invitrogen) and separated using NuPage bis-tris precast polyacrylamide gels (Invitrogen). Proteins were transferred to a PVDF membrane (BioRad). Western blot was performed as described in [32 (link)]. Affinity-purified anti-CYP51 antibodies (Genescript) and anti-GAPDH antibody (from Paul Michels, Université catholique de Louvain, Bruxelles) were used at 1:5,000 dilution. The secondary antibody was a 1:5,000 dilution of peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare). All proteins were visualized using SuperSignal West Pico Chemoluminescent Substrate (Thermo Scientific). Proteins expression levels were quantified with Image J program, normalizing CYP51 levels to GAPDH levels.