Anti-Histone H3 antibody [EPR21228] - Nuclear Marker

• ChIP

Lab

ChIP - Anti-Histone H3 antibody [EPR21228] - Nuclear Marker (AB213257)

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).

• PepArr

Lab

Peptide Array - Anti-Histone H3 antibody [EPR21228] - Nuclear Marker (AB213257)

ab213257 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 ab213257 was 1/500 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 a full list of all peptides and information on the position of each peptide in the diagram, can be downloaded from the Support and downloads tab.

• WB

Lab

Western blot - Anti-Histone H3 antibody [EPR21228] - Nuclear Marker (AB213257)

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.

All lanes:

Western blot - Anti-Histone H3 antibody [EPR21228] - Nuclear Marker (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

Predicted band size: 15 kDa

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Exposure time: 12min