Anti-KDM4B / JMJD2B antibody [EPR25240-41]

• IHC

Supplier Data

Immunohistochemistry - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling KDM4B / JMJD2B with ab305056 at 1/200 dilution (2.575 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in human breast carcinoma. The section was incubated with ab305056 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling KDM4B / JMJD2B with ab305056 at 1/200 dilution (2.575 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in human breast. The section was incubated with ab305056 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Immunohistochemical analysis of paraffin-embedded cell pellets labeling KDM4B / JMJD2B with ab305056 at 1/200 dilution (2.575 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on (A) parental HAP1 cell pellet, (B) SW480 cell pellet, and (C) HEK-293 cell pellet, no staining on (D) KDM4B knockout HAP1 cell pellet. The section was incubated with ab305056 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized wild-type HAP1 cells labeling KDM4B / JMJD2B with ab305056 at 1/50 dilution (10.3 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing nuclear staining in parental HAP1 cell line, and no staining in KDM4B KO HAP1 cell line. 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 (2.5 ug/ml) (Red). 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 dilution (2 ug/ml).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SW480 (human colorectal adenocarcinoma epithelial cell) cells labeling KDM4B / JMJD2B with ab305056 at 1/50 dilution (10.3 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing nuclear and weak cytoplasmic staining in SW480 cell line. 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 (2.5 ug/ml) (Red). 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 dilution (2 ug/ml).

• WB

Lab

Western blot - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Western blot : Anti-KDM4B antibody [EPR25240-41] (ab305056) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab305056 was shown to bind specifically to KDM4B. A band was observed at 135 kDa in wild-type A549 cell lysates with no signal observed at this size in KDM4B knockout cell line. To generate this image, wild-type and KDM4B knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (ab305056) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human KDM4B Knockout A549 cell line (<a href='/ko/products/cell-lines/human-kdm4b-knockout-a549-cell-line-ab324212'>ab324212</a>) at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 122 kDa

Observed band size: 135 kDa

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• WB

Supplier Data

Western blot - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (AB305056)

Buffer/dilution : 5% NFDM/TBST Exposure : 158 seconds Performed under reducing conditions. In Western blot, ab305056 was shown to bind specifically to KDM4B/JMJD2B. A band was observed at 160 kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in KDM4B/JMJD2B knockout cell line.

All lanes:

Western blot - Anti-KDM4B / JMJD2B antibody [EPR25240-41] (ab305056) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

KDM4B / JMJD2B knockout HAP1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 160 kDa

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Exposure time: 158s