Recombinant Anti-HDAC1 antibody [EPR23847-170] (ab280198)

ab280198 Anti-HDAC1 antibody [EPR23847-170] was shown to specifically react with HDAC1 in wild-type HAP1 cells. Loss of signal was observed when knockout cell line (knockout cell lysate) was used. Wild-type and HDAC1 knockout samples were subjected to SDS-PAGE. ab280198 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Chromatin was prepared from K-562 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab280198 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

HDAC1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab280198 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280198 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug

Lane 2: ab280198 IP in NIH/3T3 whole cell lysate 10 ug

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280198 in NIH/3T3 whole cell lysate

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

Exposure time: 1 second.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling HDAC1 with ab280198 at 1/5000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in HeLa cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) 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 dilution.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling HDAC1 with ab280198 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human tonsil (PMID:23109994). The section was incubated with ab280198 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.

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

The observed MW is consistent with what has been described in the literature (PMID:24551070).

Exposure time: 15 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixe,d 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling HDAC1 with ab280198 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

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

The observed MW is consistent with what has been described in the literature (PMID: 24551070).

Exposure time: 37 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling HDAC1 with ab280198 at 1/5000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) 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 dilution.

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

This antibody has no cross-reaction with human HDAC2.

These recombinant proteins were made in house. These two recombinant proteins were expressed from E.coli expression systems.

Exposure time: 10 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling HDAC1 with ab280198 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling HDAC1 with ab280198 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human liver (PMID:18264140). The section was incubated with ab280198 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

HDAC1 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab280198 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280198 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab280198 IP in HeLa whole cell lysate 10 ug

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280198 in HeLa whole cell lysate

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

Exposure time: 1 second.

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

The observed MW is consistent with what has been described in the literature (PMID:24551070).

Exposure time: 37 seconds.

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 2.5 x 10⁵ K562 (Human chronic myelogenous leukemia lymphoblast cell line) cells and 5 µg of ab280198  [EPR23847-170]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.

Additional screenshots of mapped reads can be downloaded here.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling HDAC1 with ab280198 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse liver. The section was incubated with ab280198 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.

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling HDAC1 with ab280198 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat liver. The section was incubated with ab280198 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.