Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Human colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab176840 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• ChIP-seq

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ChIP-sequencing - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 μg of chromatin and 4 μg of Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA. Additional screenshots of mapped reads can be downloaded here.

• ChIP

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ChIP - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) 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 ab176840 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Rat colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Histone H3.3 with ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on mouse stomach tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa,47 kDa,58 kDa

Observed band size: 15 kDa,59 kDa

false

Exposure time: 3min

• WB

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Western blot - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Blocking experiment by pre-incubating Histone H3.3 peptide with the antibody in lanes 2 and 5 and Histone H3.1 peptide in lanes 3 and 6 to show the antibody specificity against Histone H3.3.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (ab176840) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg with Histone H3.3 peptide

Lane 3:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg with Histone H3.1 peptide

Lane 4:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Lane 5:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg with Histone H3.3 peptide

Lane 6:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg with Histone H3.1 peptide

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

Exposure time: 3min

• Dot

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Dot Blot - Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade (AB176840)

Dot blot analysis of Histone H3.3 peptide (Lane 1), and Histone H3.1 peptide (Lane 2), labeled using ab176840 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer : 5% NFDM/TBST.