Anti-HDAC2 antibody [Y461]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [Y461] (AB32117)

Immunohistochemical analysis of paraffin-embedded fixed (A) Wild-type HEK293T (human embryonic kidney epithelial cell) cell pellet. (B) HDAC2 knockout HEK293T (ab266590) cell pellet staining HDAC2 with ab32117 at 1/10000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Counter-staining used was hematoxylin. Positive staining on (A) Wild-type HEK293T cell pellet, no staining on HDAC2 knockout HEK293T (ab266590) cell pellet. The section was incubated with ab32117 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody [Y461] (AB32117)

Immunocytochemistry/ Immunofluorescence analysis of Wild-type HEK293T/HDAC2 KO HEK293T (HDAC2 knockout human embryonic kidney epithelial cell) (ab266590) cells labeling HDAC2 with ab32117 at 1/200 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody at 1/1000 dilution (2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (2.5 µg/ml). DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control. Confocal image showing nuclear staining in Parental HEK293T cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [Y461] (AB32117)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue sections labeling HDAC2 with Purified ab32117 at 1 : 1500 dilution (0.071 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HDAC2 antibody [Y461] (AB32117)

Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling HDAC2 with Purified ab32117 at 1 : 20 dilution (5 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody [Y461] (AB32117)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HDAC2 with Purified ab32117 at 1 : 50 dilution (2.1 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody [Y461] (AB32117)

ab32117 staining HDAC2 in wild-type HAP1 cells (top panel) and HDAC2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32117 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [Y461] (AB32117)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling HDAC2 with Purified ab32117 at 1 : 1500 dilution (0.071 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• IP

Lab

Immunoprecipitation - Anti-HDAC2 antibody [Y461] (AB32117)

Purified ab32117 at 1 : 20 dilution (0.5μg) immunoprecipitating HDAC2 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg.
Lane 2 (+) : ab32117 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32127 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)(ab131366) (1 : 5000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : kDa

All lanes:

Immunoprecipitation - Anti-HDAC2 antibody [Y461] (ab32117)

Predicted band size: 55 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [Y461] (AB32117)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat testis tissue sections labeling HDAC2 with Purified ab32117 at 1 : 1500 dilution (0.071 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC2 antibody [Y461] (AB32117)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue sections labeling HDAC2 with Purified ab32117 at 1 : 1500 dilution (0.071 μg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• WB

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Western blot - Anti-HDAC2 antibody [Y461] (AB32117)

All lanes:

Western blot - Anti-HDAC2 antibody [Y461] (ab32117) at 1/10000 dilution

Lane 1:

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

Lane 2:

Mouse brain lysate at 20 µg

Lane 3:

Rat brain 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 1/20000 dilution

Predicted band size: 55 kDa

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• WB

Lab

Western blot - Anti-HDAC2 antibody [Y461] (AB32117)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : HDAC2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : A431 whole cell lysate (20 μg)
Lane 4 : Hela whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32117 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32117 was shown to specifically react with HDAC2 when HDAC2 knockout samples were used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab32117 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 and 1/10000 respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HDAC2 antibody [Y461] (ab32117)

Predicted band size: 55 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HDAC2 antibody [Y461] (AB32117)

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 ab32117, 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 [Y461] (AB32117)

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 ab32117, 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 [Y461] (AB32117)

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 5 µg of ab32117 [Y461] . 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 [Y461] (AB32117)

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

All lanes:

Western blot - Anti-HDAC2 antibody [Y461] (ab32117) at 1/1000 dilution

Lane 1:

GH3 (rat pituitary epithelial cell) whole cell lysate at 20 µg

Lane 2:

L6 (rat skeletal muscle myoblast) whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 4:

AR42J (rat pancreatic tumor epithelial cell) whole cell lysate at 20 µg

Lane 5:

2.4G2 (rat B cell lymphoma B lymphocyte) 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: 60 kDa

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Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-HDAC2 antibody [Y461] (AB32117)

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

All lanes:

Western blot - Anti-HDAC2 antibody [Y461] (ab32117) at 1/1000 dilution

Lane 1:

HT-22 (mouse hippocampal neuronal cell) whole cell lysate at 20 µg

Lane 2:

SW10 (mouse neuronal Schwann cell) whole cell lysate at 20 µg

Lane 3:

bEnd.3 (mouse brain endothelioma) 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: 60 kDa

false

Exposure time: 26s