Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3.3 mutated G34W and Histone H3.3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab272691 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are located in the first kb of the transcribed region.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

All lanes : Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 15 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

Flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized HEK-293T (Human embryonic kidney epithelial cell) transfected with myc tagged Histone H3.3 WT construct (Left panel) and myc-tagged Histone H3.3 G34W construct (Right panel) cells labelling Histone H3.3(mutated G34 W) with ab272691 at 1/50 dilution (1µg). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells labelling Histone H3.3(mutated G34 W) with ab272691 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with Histone H3.3 G34W-Myc plasmid, while no staining in HEK-293T cells transfected with H3.3 WT -Myc plasmid. Myc-Tag Mouse mAb (Alexa Fluor^® 647) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3(mutated G34 W) with ab272691 at 1/250 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human giant cell tumor of bone (PMID: 29757500). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3.3(mutated G34 W) with ab272691 at 1/250 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP).

Negative control: No staining in human chondroblastoma (PMID: 29757500).

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Dot blot analysis of Histone H3.3 (mutated G34 W) labeled with ab272691 at 1/1000 dilution.

Lane 1: Histone H3.3 H3G34W peptide (aa28-37).
Lane 2: Histone H3.3 H3G34W peptide (aa33-43).
Lane 3: Histone H3.3 H3G34W peptide (aa28-43).
Lane 4: Histone H3.3 WT peptide (aa28-43).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.