Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3(K27 M) and Histone H3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min. 
The ChIP was performed with 25 µg of chromatin, 2 µg of ab190631 (red), 2 µg of ab213204 (red) (bottom panel, served as internal control) 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 (mutated K27 M) antibody [EPR18340] (ab190631) at 1/1000 dilution.

Lane 1: HEK-293 transfected with Histone H3.1 (K27M) expression vector containing a myc-His-tag, whole cell lysate, 10 ug.

Lane 2: HEK-293 transfected with Histone H3(WT) expression vector containing a myc-His-tag, whole cell lysate, 10 ug.

Secondary (all lanes): ab97051 at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 18 kDa.

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

Exposure time: 6 seconds.

All lanes: Anti-Histone H3 (mutated K27 M) antibody [EPR18340] (ab190631) at 1/1000 dilution.

Lane 1: HEK-293 transfected with Histone H3.3 (K27M) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

Secondary (all lanes): ab97051 at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 18 kDa.

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

Exposure time: 3 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling Histone H3 (K27 mutated to M) with ab190631 at 1/5000 (0.2 ug/ml) dilution, followed by ab190631 anti- H3(K27 mutated to M) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with myc-tagged H3 (K27 mutated to M) expression vector is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Ab190631 anti- H3(K27 mutated to M) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

All lanes: Anti-Histone H3 (mutated K27 M) antibody [EPR18340] (ab190631) at 1/1000 dilution.

Lane 1: His-tagged recombinant histone H3 K27M protein, 0.01 ug

Lane 2: His-tagged recombinant wild type histone H3 protein, 0.01 ug

Secondary (all lanes): ab97051 at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 15 kDa.

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

Exposure time: 15 seconds.

All lanes: Anti-Histone H3 (mutated K36 M) antibody [EPR23614-91] (ab256384) at 1/1000 dilution.

Lane 1: HEK-293 transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

Lane 2: HEK-293 transfected with Histone H3.3 K36M (mutated) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug.

Secondary (all lanes): ab97051 at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 20 kDa.

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

Exposure time: 26 seconds

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T transfected with myc tagged Histone H3 construct (Left) or myc tagged Histone H3 K36M construct (Right) cells labelling Histone H3 (mutated K36 M) with ab256384 at 1/500 compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293 cells labelling Histone H3 (mutated K36 M) with ab256384 at 1/50 dilution, followed by a secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HEK-293 cell line transfected with myc-tagged Histone H3 K36M expression vector. 2233S Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 647 Conjugate) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3 (mutated K36 M) with ab256384 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Negative control: No staining in human giant cell tumor of bone (PMID: 29757500). Counterstained with Hematoxylin.

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

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

All lanes: Anti-Histone H3 (mutated K36 M) antibody [EPR23614-91] (ab256384) at 1/1000 dilution.

Lane 1: HEK-293 transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug

Lane 2: HEK-293 transfected with Histone H3.3 K36M (mutated) expression vector containing a myc-His-tag®, whole cell lysate, 10 ug.

Secondary (all lanes): ab97051 at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 20 kDa.

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

Exposure time 62 seconds.

Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3 (mutated K36 M) with ab256384 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human chondroblastoma (PMID: 29757500). Counterstained with Hematoxylin.

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

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

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 G34 W) 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, 40 ug.

Lane 2: HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate, 40 ug.

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

Predicted MW: 15 kDa.

Observed MW: 20 kDa.

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.

Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3.3 mutated G34V and Histone H3.3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab254401 (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 G34 V) antibody [EPR23520-5] - ChIP Grade (ab254401 at 1/1000 dilution.

Lane 1: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag®, whole cell lysate, 20 ug

Lane 2: HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate, 20 ug.

Secondary (all lanes): Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 20 kDa.

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

Exposure time: 10 seconds.

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 H3G34V construct (Right panel) cells labelling Histone H3.3(mutated G34 V) with ab254401 at 1/500 dilution (0.1µg). 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 V) with ab254401 at 1/1000 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 H3G34V-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 V) with ab254401 at 1/1000 dilution (0.542 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human giant cell tumor of bone. (PMID: 29241742). The section was incubated with ab254401 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3.3(mutated G34 V) with ab254401 at 1/1000 dilution (0.542 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control: No staining on human chondroblastoma (PMID: 29241742).

The section was incubated with ab254401 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Dot blot analysis of Histone H3.3 (mutated G34 V) labeled with ab254401 at 1/1000 dilution.

Lane 1: Histone H3.3 H3G34V peptide (aa28-40).
Lane 2: Histone H3.3 H3G34V peptide (aa26-38).
Lane 3: Histone H3.3 WT peptide (aa26-40).

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.

Chromatin was prepared from HEK-293T transfected with Histone H3.3(mutated G34 R) and HEK-293T transfacted with Histone H3.3(WT) cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab254402 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).

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

Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human giant cell tumor of bone (PMID: 29241742). The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

All lanes:  Anti-Histone H3.3 (mutated G34 R) antibody [EPR23519-91] - ChIP Grade (ab254402) at 1/1000 dilution.

Lane 1: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containi a myc-His-tag®, whole cell lysate, 20 ug.

Lane 2:  HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate, 20 ug.

Lane 3: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag®, whole cell lysate, 20 ug.

Lane 4: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containngi a myc-His-tag®, whole cell lysate, 20 ug.

Secondary (all lanes): Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051), 1/100000 dilution.

Predicted MW: 15 kDa.

Observed MW: 20 kDa.

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

Exposure time: 26 seconds. 

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 expression vector) (Left) or myc-tagged Histone H3.3 G34R expression vector (Right) cells labelling Histone H3.3 (mutated G34 R) with ab254402 at 1/5000 dilution (0.01ug) (Both panels). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Dot blot analysis of Histone H3.3 (mutated G34 R) labeled with ab254402 at 1/1000 dilution.

Lane 1: Histone H3.3 H3G34R peptide (aa28-40).
Lane 2: Histone H3.3 H3G34R peptide (aa26-36).
Lane 3: Histone H3.3 WT peptide (aa26-40).

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.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human testis. The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.