Anti-Histone H3 antibody - Nuclear Marker and ChIP Grade(AB1791)

Chromatin from Xenopus laevis oocytes was prepared according to the Abcam X-ChIP protocol.

Oocytes were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 mg of chromatin, 3 mg of ab7834 (anti-H3, light blue) and 3 μg of ab1791 (anti-H3, dark blue), and 20 ml of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow).

The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

Histone H3 - ChIP Grade was immunoprecipitated using 0.5mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 μg of Rabbit polyclonal to and 50 μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.

Proteins were eluted by addition of 40 μl SDS loading buffer and incubated for 10 minutes at 70°C; 10 μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1791.

Secondary Antibody: Mouse anti-rabbit HRP light chain (HRP) (ab99697).

Band: 15kDa; Histone H3 - ChIP Grade

All lanes:

Immunoprecipitation - Anti-Histone H3 antibody - Nuclear Marker and ChIP Grade (ab1791)

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa

Exposure time: 30s

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 2% Bovine Serum Albumin before being incubated with ab1791 overnight at 4°C.

Goat Anti-Rabbit IgG H&L (HRP) (ab6721) secondary antibody was used for detection.

Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H3 antibody - Nuclear Marker and ChIP Grade (ab1791) at 1/1000 dilution

Lanes 1 and 4:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lanes 2 and 5:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 20 µg

Lanes 3 and 6:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa

Observed band size: 17 kDa

Exposure time: 10s

ab1791 staining Histone H3 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1791 at 0.1 μg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).