Anti-HDAC1 + HDAC2 + HDAC3 + HDAC8 抗体 [RM1172] (ab319059)

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

In Western blot, ab319059 was shown to bind specifically to HDAC1, HDAC2, HDAC3, HDAC8.  Target of interest was observed at 55kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in HDAC1 knockout cell line (lane 2).Target of interest was observed at 55kDa in wild-type 293T cell lysates (lane 3) with no signal observed at this size in HDAC2 knockout cell line (lane 4), (lane 4, knockout cell line ab266590 / knockout cell lysate ab256939).Target of interest was observed at 42kDa in wild-type HAP1 cell lysates (lane 5) with no signal observed at this size in HDAC8 knockout cell line (lane 6).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

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

Exposure time: Lanes 1-4: 10 seconds Lanes 5-9: 48 seconds

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

The identity of the lower MW band at approximately 35 kDa is unknown. 

Lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lanes 3-6 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

HDAC1 + HDAC2 + HDAC3 + HDAC8 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab319059 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab319059 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2: ab319059 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab319059 in HeLa whole cell lysate

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

Exposure time: 15 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/1000 (0.502 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in Hela 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 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/1000 (0.502 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in RAW 264.7 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 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C6 (rat glial tumor glial cell) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/1000 (0.502 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

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 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab319059 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The section was incubated with ab319059 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat colon. The section was incubated with ab319059 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/500 dilution (0.1ug) / Magenta  compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/500 dilution (0.1ug) / Magenta  compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) cells labelling HDAC1 + HDAC2 + HDAC3 + HDAC8 with ab319059 at 1/500 dilution (0.1ug) / Magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.