Anti-KDM2A antibody [EPR18602]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KDM2A antibody [EPR18602] (AB191387)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling KDM2A with ab191387 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Jurkat cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows : -
-ve control 1 : ab191387 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM2A antibody [EPR18602] (AB191387)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling KDM2A with ab191387 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells of human colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KDM2A antibody [EPR18602] (AB191387)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling KDM2A with ab191387 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows : -
-ve control 1 : ab191387 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM2A antibody [EPR18602] (AB191387)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling KDM2A with ab191387 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells of mouse colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM2A antibody [EPR18602] (AB191387)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling KDM2A with ab191387 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells of rat colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-KDM2A antibody [EPR18602] (AB191387)

KDM2A was immunoprecipitated from 1mg of mouse brain whole cell lysate with ab191387 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab191387 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Mouse brain whole cell lysate 10μg (Input).

Lane 2 : ab191387 IP in Mouse brain whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab191387 in Mouse brain whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-KDM2A antibody [EPR18602] (ab191387)

Predicted band size: 133 kDa

false

• WB

Lab

Western blot - Anti-KDM2A antibody [EPR18602] (AB191387)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : KDM2A knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Hela whole cell lysate (20 μg)
Lane 4 : Jurkat whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab191387 observed at 133 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab191387 was shown to specifically react with KDM2A in wild-type HAP1 cells. No band was observed when KDM2A knockout cells were examined. Wild-type and KDM2A knockout samples were subjected to SDS-PAGE. ab191387 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1.313 ug/ml and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-KDM2A antibody [EPR18602] (ab191387)

Predicted band size: 133 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM2A antibody [EPR18602] (AB191387)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 8 seconds; Lane 3 : 15 seconds.

This antibody recognizes two isoforms. The predicted MW are 133kDa and 90kDa respectively.

All lanes:

Western blot - Anti-KDM2A antibody [EPR18602] (ab191387) 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 cell line from peripheral blood) whole cell lysate at 20 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) 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: 133 kDa

Observed band size: 133 kDa,90 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM2A antibody [EPR18602] (AB191387)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM2A antibody [EPR18602] (ab191387) at 1/5000 dilution

All lanes:

Human brain lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 133 kDa

Observed band size: 133 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-KDM2A antibody [EPR18602] (AB191387)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 3 : 3 minutes; Lane 2 : 30 seconds.

All lanes:

Western blot - Anti-KDM2A antibody [EPR18602] (ab191387) at 1/1000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Mouse testis lysate at 10 µg

Lane 3:

Mouse brain lysate at 10 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: 133 kDa

Observed band size: 133 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM2A antibody [EPR18602] (AB191387)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 15 seconds; Lane 3 : 8 seconds; Lane 4 : 30 seconds.

All lanes:

Western blot - Anti-KDM2A antibody [EPR18602] (ab191387) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

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

Lane 4:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 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: 133 kDa

Observed band size: 133 kDa

false