ab174992 staining Histone H3 (citrulline R2) in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab174992 at 5µg/ml and ab7291 (anti beta Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat anti-rabbit AlexaFluor®488 secondary antibody (ab150081) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
All lanes : Anti-Histone H3 (citrulline R2) antibody (ab174992) at 1 µg/mlLane 1 : HL60 (Human promyelocytic leukemia cell line) Whole Cell LysateLane 2 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Lonophore treated Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.