Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• ChIP

Supplier Data

ChIP - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• ChIP

Lab

ChIP - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

Chromatin was prepared from HEK-293T transfacted with Histone H3.3(mutated G34 R) and 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).

• ChIP

Supplier Data

ChIP - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• WB

Lab

Western blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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), 1/100000 dilution.

Predicted MW : 15 kDa.

Observed MW : 20 kDa.

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

Exposure time : 10 seconds.

All lanes:

Western blot - Anti-Histone H3.3 (mutated G34V) antibody [EPR23520-5] - ChIP Grade (<a href='/en-us/products/primary-antibodies/histone-h33-mutated-g34v-antibody-epr23520-5-chip-grade-ab254401'>ab254401</a>) 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 at 20 µg

Lane 2:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 15 kDa

Observed band size: 20 kDa

false

• WB

Lab

Western blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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

Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R 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.

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.

All lanes:

Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (<a href='/en-us/products/primary-antibodies/histone-h33-mutated-g34r-antibody-epr23519-91-chip-grade-ab254402'>ab254402</a>) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

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

Lane 4:

HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 15 kDa

Observed band size: 20 kDa

false

• WB

Lab

Western blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

All lanes:

Western blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (<a href='/en-us/products/primary-antibodies/histone-h33-mutated-g34w-antibody-epr23581-39-chip-grade-ab272691'>ab272691</a>) 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 at 40 µg

Lane 2:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 20 kDa

false

• Dot

Supplier Data

Dot Blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• Dot

Supplier Data

Dot Blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.

• Dot

Lab

Dot Blot - Human Histone H3.3 (Mutated G34W, G34V, G34R) Antibody Panel (AB274410)

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.