Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25µg of chromatin, 5µg of ab213257 (red), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

All lanes : Anti-Histone H3 antibody [EPR21228] (ab213257) at 1 µg/ml

Lane 1 : Calf Thymus Histone lysate at 0.5 µg
Lane 2 : Hela whole cell lysate at 20 µg
Lane 3 : HeLa histone preparation nuclear lysate at 10 µg
Lane 4 : NIH3T3 whole cell lysate at 10 µg
Lane 5 : NIH3T3 histone preparation nuclear lysate at 10 µg
Lane 6 : Histone H2B recombinant protein at 0.1 µg
Lane 7 : Histone H3.1 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: 17 kDa


Exposure time: 12 minutes

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 40 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 ab213257 overnight at 4°C. Antibody binding was detected using a goat anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.