Anti-HDAC2 antibody [EPR20117]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human prostate hyperplasia tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on luminal epithelial cells of human prostate hyperplasia; negative staining on basal cells.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on tumor cells of human breast cancer 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) Ready to use.

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-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on lymphocytes of human tonsil 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) Ready to use.

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-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on human testis 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) Ready to use.

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-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human synovial sarcoma tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on human synovial sarcoma 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) Ready to use.

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-HDAC2 antibody [EPR20117] (AB219053)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293 (Human epithelial cell line from embryonic kidney) cells labeling HDAC2 with ab219053 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HEK-293 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded human prostate cancer tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear Nuclear staining on tumor cells of prostate cancer; weak or negative staining on basal cells.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

• IP

Supplier Data

Immunoprecipitation - Anti-HDAC2 antibody [EPR20117] (AB219053)

HDAC2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab219053 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab219053 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).

Lane 2 : ab219053 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab219053 in HeLa whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-HDAC2 antibody [EPR20117] (ab219053)

Predicted band size: 55 kDa

Observed band size: 55 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody [EPR20117] (AB219053)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HDAC2 with ab219053 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on mouse colon 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) Ready to use.

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HDAC2 antibody [EPR20117] (AB219053)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling HDAC2 with ab219053 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [EPR20117] (AB219053)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling HDAC2 with ab219053 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Nuclear staining on rat spleen 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) Ready to use.

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

• WB

Lab

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Lanes 1- 2 : Merged signal (red and green). Green - ab219053 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab219053 was shown to react with HDAC2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266589 (knockout cell lysate ab256938) was used. Wild-type HEK-293T and HDAC2 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab219053 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HDAC2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human HDAC2 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hdac2-knockout-hek-293t-cell-line-ab266589'>ab266589</a>)

Predicted band size: 55 kDa

Observed band size: 55 kDa

false

• WB

Supplier Data

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) 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: 55 kDa

Observed band size: 55 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2 : 15 seconds; Lane 3 : 2 seconds.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/5000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 55 kDa

Observed band size: 55 kDa

false

• WB

Lab

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Lanes 1 - 4 : Merged signal (red and green). Green - ab219053 observed at 55 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab219053 was shown to specifically react with HDAC2 in wild type cells as signal was lost in HDAC2 knockout cells. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab219053 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HDAC2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A431 whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 55 kDa

false

• WB

Lab

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Lanes 1-2 : Merged signal (red and green). Green - ab219053 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

ab219053 Anti-HDAC2 antibody [EPR20117] was shown to specifically react with HDAC2 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266588 (knockout cell lysate ab256937) was used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab219053 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HDAC2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human HDAC2 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hdac2-knockout-hek-293t-cell-line-ab266588'>ab266588</a>)

Predicted band size: 55 kDa

Observed band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Lanes 1-2 : Merged signal (red and green). Green - ab219053 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

ab219053 Anti-HDAC2 antibody [EPR20117] was shown to specifically react with HDAC2 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266590 (knockout cell lysate ab256939) was used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab219053 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HDAC2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human HDAC2 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hdac2-knockout-hek-293t-cell-line-ab266590'>ab266590</a>)

Predicted band size: 55 kDa

Observed band size: 60 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HDAC2 antibody [EPR20117] (AB219053)

CUT&RUN profiling with HDAC2 antibody demonstrates robust genome-wide enrichment in wild-type (WT) cells, which is markedly diminished in HDAC2 knockout (KO) cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with HDAC2 antibody (Abcam ab219053, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab266590) cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramнrez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to HDAC2 WT, with red indicating high localized enrichment and blue denoting background. Validated antibodies show genome-wide enrichment above IgG background consistent with HDAC2 binding in WT cells and near complete loss of signal in KO cells.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HDAC2 antibody [EPR20117] (AB219053)

CUT&RUN profiling with HDAC2 antibody reveals the expected genomic enrichment pattern in wild-type (WT) cells, which is substantially reduced in HDAC2 knockout (KO) cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with HDAC2 antibody (Abcam ab219053, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab266590) cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HDAC2 antibody [EPR20117] (AB219053)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 K-562 (Human chronic myelogenous leukemia lymphoblast) cells and 5µg of ab219053 [EPR20117]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Supplier Data

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 15 seconds; Lane 3 : 30 seconds; Lane 4/5 : 3 seconds; Lane 6/7 : 1 second.

Lanes 1 - 5:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lanes 6 - 7:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/5000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat heart lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 µg

Lane 6:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 7:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 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: 11 kDa,55 kDa

Observed band size: 55 kDa

false

• WB

Supplier Data

Western blot - Anti-HDAC2 antibody [EPR20117] (AB219053)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HDAC2 antibody [EPR20117] (ab219053) at 1/1000 dilution

Lane 1:

His-tagged human HDAC2 recombinant protein (aa339-488) at 0.01 µg

Lane 2:

His-tagged human HDAC1 recombinant protein (aa1-482) at 0.01 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: 55 kDa

Observed band size: 22 kDa

false

Exposure time: 1s