Anti-Histone H2B antibody [IGX4228R-1]

• ChIP

Lab

ChIP - Anti-Histone H2B antibody [IGX4228R-1] (AB213225)

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 ab213225 (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 H2B antibody [IGX4228R-1] (AB213225)

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 ab213225 (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.

• PepArr

Lab

Peptide Array - Anti-Histone H2B antibody [IGX4228R-1] (AB213225)

ab213225 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 ab213225 was 1/5000 dilution, the secondary used was Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated 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.

• Dot

Lab

Dot Blot - Anti-Histone H2B antibody [IGX4228R-1] (AB213225)

Dot blot analysis using ab213225 at 1/1000 dilution and secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution. Blocking and diluting buffer was 5% NFDM/TBST. Lane 1 : Human Histone H2B peptide Lane 2 : Human Histone H1.4 (methyl K25) peptide Exposure time : 180 seconds

• WB

Lab

Western blot - Anti-Histone H2B antibody [IGX4228R-1] (AB213225)

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 ab213225 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H2B antibody [IGX4228R-1] (ab213225) at 0.25 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 H2B Recombinant Protein at 0.1 µg

Lane 7:

Histone H3 Recombinant Protein at 0.1 µg

Secondary

All lanes:

Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

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Exposure time: 1min