Anti-KDM4D / JMJD2D antibody [EPR25241-24]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (AB289667)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling KDM4D / JMJD2D with ab289667 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line. The nuclear counterstain is DAPI (Blue). Tubulin is labeled using ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (AB289667)

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell line) cells labeling KDM4D / JMJD2D with ab289667 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081)at 1/2000 dilution was used as the secondary antibody.

• IP

Lab

Immunoprecipitation - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (AB289667)

KDM4D / JMJD2D was immunoprecipitated from HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab289667 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab289667 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab289667 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289667 in HeLa whole cell lysate.

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

All lanes:

Immunoprecipitation - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (ab289667)

Predicted band size: 59 kDa

Observed band size: 60 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (AB289667)

5% NFDM/TBST was used as a dilution and blocking buffer.

Low expression : ovary, spleen (PMID : 21293030).

All lanes:

Western blot - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (ab289667) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human ovary tissue lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG (Merck DC03L) at 1/2000 dilution

Predicted band size: 59 kDa

Observed band size: 60 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (AB289667)

Lysates were made freshly and used in the WB test immediately to minimize protein degradation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM4D / JMJD2D antibody [EPR25241-24] (ab289667) at 1/1000 dilution

All lanes:

HeLa (human cervix adenocarcinoma epithelial 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 1/100000 dilution

Predicted band size: 59 kDa

Observed band size: 60 kDa

false

Exposure time: 3min