Anti-HDAC2 antibody [EPR20117] - BSA and Azide free

• WB

Lab

Western blot - Anti-HDAC2 antibody [EPR20117] - BSA and Azide free (AB251561)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/hdac2-antibody-epr20117-ab219053'>ab219053</a>) 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

Unknown

Western blot - Anti-HDAC2 antibody [EPR20117] - BSA and Azide free (AB251561)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/hdac2-antibody-epr20117-ab219053'>ab219053</a>) 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

• WB

Lab

Western blot - Anti-HDAC2 antibody [EPR20117] - BSA and Azide free (AB251561)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/hdac2-antibody-epr20117-ab219053'>ab219053</a>) 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

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HDAC2 antibody [EPR20117] - BSA and Azide free (AB251561)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB251561)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB251561)

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. This data was developed using the same antibody clone in a different buffer formulation (ab219053).