All lanes : Anti-Histone H2B (crotonyl K5) antibody (ab177139) at 1 µg/mlLane 1 : HeLa Nuclear Prep (0.5% Triton X-100 insoluble fraction) at 10 µgLane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 3 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µgSecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ab177139 staining Histone H2B (crotonyl K5) 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 ab177139 at 5μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150081) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.Negative controls: 1– Rabbit primary 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.