Rabbit anti histone h3 antibody

Streptavidin Fluoresbrite YG Carboxylate Microspheres (Polyscience) were coated with biotinylated Protein L (Genescript) in phosphate-buffered saline (PBS) with sodium chloride and bovine serum albumin. Protein L-coated beads were then incubated with blood from WT and Ncf1** mice at 37 °C, 5% CO₂ for 1 h for uptake. After removal of erythrocytes by hypotonic lysis, remaining cells were taken up in Opti-MEM I + GlutaMAX-I (Gibco) supplemented with 10% FCS and 1% Penicillin/Streptomycin and incubated at 37 °C, 5% CO₂ for another 0–4 h. After washing with PBS, pelleted cells were lysed with Laemmli buffer (Sigma) for 10 min at 95 °C. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 8–16% gradient gels (ServaGel TG PRiME, Serva). The protein material was then blotted to polyvinylidene difluoride membranes (Immobilon, Merck). Membranes were blocked for 1 h with Tris-buffered saline (TBS)/5% milk powder and incubated for 1 h with Streptavidin-HRP (Thermo Scientific Cat No. #N100). Finally, bands were visualised by enhanced chemiluminescence (ThermoScientific). Densitometric analysis was performed using ImageJ software (Rasband, WS, ImageJ, NIH, Bethesda, USA). Histone H3 served as a loading control and was visualised using a rabbit anti-histone H3 antibody (ab1791 Abcam). Remaining Protein L content was normalized to histone H3 in every lane.