Anti-Histone H4 antibody [IGX4696H] - ChIP Grade

• ChIP

Lab

ChIP - Anti-Histone H4 antibody [IGX4696H] - ChIP Grade (AB213291)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab213291 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

• ChIP

Lab

ChIP - Anti-Histone H4 antibody [IGX4696H] - ChIP Grade (AB213291)

Chromatin was prepared from NIH3T3 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab213291 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

• WB

Lab

Western blot - Anti-Histone H4 antibody [IGX4696H] - ChIP Grade (AB213291)

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 ab213291 overnight at 4°C. Antibody binding was detected using an anti-human antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H4 antibody [IGX4696H] - ChIP Grade (ab213291) at 0.2 µg/mL

Lane 1:

CTH (Calf Thymus Histone) at 0.5 µg

Lane 2:

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

Lane 3:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

Lane 4:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 5:

NIH 3T3 (Mouse embryonic fibroblast cell line) Nuclear Lysate at 10 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-Human IgG (H+L) at 1/10000 dilution

true

Exposure time: 4min

• PepArr

Lab

Peptide Array - Anti-Histone H4 antibody [IGX4696H] - ChIP Grade (AB213291)

ab213291 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). The concentration of ab213291 was 1/5000 dilution, the secondary used was Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 at 1/50000 dilution. Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Product Protocol section.