Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. ChIP was performed with 25 microgram of chromatin, 2 microgram of ab223875 (red), and 20 microliter of Protein A/G sepharose beads. No antibody was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region

All lanes : Anti-Histone H4 antibody [EPR21213] - ChIP Grade (ab223875) at 1 µg/ml

Lane 1 : CTH (calf Thymus Histone) at 0.5 µg
Lane 2 : HeLa Whole Cell Lysate at 20 µg
Lane 3 : HeLa Nuclear Cell Lysate at 20 µg
Lane 4 : NIH 3T3 Whole Cell Lysate at 20 µg
Lane 5 : NIH 3T3 Nuclear Lysate at 20 µg
Lane 6 : Histone H2A Recombinant Protein at 0.1 µg
Lane 7 : Histone H4 Recombinant Protein at 0.1 µg

Secondary
All lanes : HRP conjugated Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 11 kDa


Exposure time: 90 seconds

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab223875 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.