Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunohistochemical analysis of paraffin-embedded human testis tissue labelling HDAC1 with ab281585 at 1/4000 (0.13 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Nuclear staining on human testis (PMID : 16960727).

The section was incubated with ab281585 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 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling HDAC1 with ab281585 at 1/50 (10.4 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). 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).

Confocal image showing mostly nuclear staining in HeLa cell line.

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma cell) cells labelling HDAC1 with ab281585 at 1/500 dilution (0.1 μg) (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.

• IP

Supplier Data

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

HDAC1 was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 10 μg with ab281585 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab281585 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat whole cell lysate 10 μg

Lane 2 : ab281585 IP in Jurkat whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab281585 in Jurkat whole cell lysate

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (ab281585)

Predicted band size: 55 kDa

false

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse hippocampus tissue labeling HDAC1 with ab281585 at 1/100 (5.55 μg/ml) dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Nuclear staining on mouse hippocampus is observed.

Secondary antibody control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling HDAC1 with ab281585 at 1/50 (10.4 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). 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).

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

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling HDAC1 with ab281585 at 1/500 dilution (0.1 μg) (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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labelling HDAC1 with ab281585 at 1/4000 (0.13 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Nuclear staining on mouse testis.

The section was incubated with ab281585 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 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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat hippocampus tissue labeling HDAC1 with ab281585 at 1/100 (5.55 μg/ml) dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Nuclear staining on rat hippocampus is observed.

Secondary antibody control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labelling HDAC1 with ab281585 at 1/4000 (0.13 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Nuclear staining on rat testis.

The section was incubated with ab281585 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 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) cells labelling HDAC1 with ab281585 at 1/100 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody at 1/1000 dilution.

Confocal image showing nuclear staining in C6 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Magenta).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) labelling HDAC1 with ab281585 at 1/50 (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).

• IP

Lab

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

HDAC1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab281585 at 30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281585 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (ab281585) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

ab281585 at 1/30 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab281585 in NIH/3T3 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 55 kDa

false

Exposure time: 3s

• IP

Lab

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

HDAC1 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell) whole cell lysate with ab281585 at 30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281585 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (ab281585) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 2:

ab281585 at 1/30 IP in C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab281585 in C6 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 55 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

False colour image of Western blot : Anti-HDAC1 antibody [RM1004] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab281585 was shown to bind specifically to HDAC1. A band was observed at 60 kDa in wild-type HAP1 cell lysates with no signal observed at this size in HDAC1 knockout cell line. To generate this image, wild-type and HDAC1 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (ab281585) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 40 µg

Lane 2:

HDAC1 knockout HAP1 cell lysate at 40 µg

Predicted band size: 55 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (AB281585)

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

Exposure time : 5 seconds

All lanes:

Western blot - Anti-HDAC1 antibody [RM1004] - Nuclear Loading Control (ab281585) at 1/1000 dilution

Lane 1:

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

Lane 2:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 4:

C6 (rat glial tumor glial cell), 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 1/50000 dilution

Predicted band size: 55 kDa

false