Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling KDM4A / JHDM3A / JMJD2A with ab191433 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining in epithelial 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.

• IP

Supplier Data

Immunoprecipitation - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

KDM4A / JHDM3A / JMJD2A was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab191433 at 1/80 dilution.

Western blot was performed from the immunoprecipitate using ab191433 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).
Lane 2 : ab191433 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab191433 in HeLa whole cell lysate.

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr18466-ab191433'>ab191433</a>)

Predicted band size: 120 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling KDM4A / JHDM3A / JMJD2A with ab191433 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining in epithelial 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.

• WB

Lab

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : DM4A / JHDM3A / JMJD2A knockout HAP1 cell lysate (20 μg)

Lane 3 : 2OS cell lysate (20 μg)

Lane 4 : C12 cell lysate (20 μg)

Lanes 1 - 4 : erged signal (red and green). Green - ab191433 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab191433 was shown to specifically react with KDM4A / JHDM3A / JMJD2A when KDM4A / JHDM3A / JMJD2A knockout samples were used. Wild-type and KDM4A / JHDM3A / JMJD2A knockout samples were subjected to SDS-PAGE. ab191433 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr18466-ab191433'>ab191433</a>)

Predicted band size: 120 kDa

false

• WB

Unknown

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure times : Lanes 1, 3-4 : 1 minute; Lane 2 : 3 minutes.

Lanes 1 - 3:

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr18466-ab191433'>ab191433</a>) at 1/5000 dilution

Lane 4:

Anti-MTHFD2 antibody [7] (<a href='/en-us/products/unavailable/mthfd2-antibody-7-ab191443'>ab191443</a>) at 1/5000 dilution

Lane 1:

Human fetal kidney at 10 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 3:

LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg

Lane 4:

U-2 OS (Human bone osteosarcoma epithelial cell line) whole call lysate at 20 µg

Secondary

Lane 1:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Lanes 2 - 3:

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

Lane 4:

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 1/5000 dilution

Predicted band size: 120 kDa

Observed band size: 150 kDa

true

• WB

Supplier Data

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] - BSA and Azide free (AB251084)

This data was developed using ab191433, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR18466] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr18466-ab191433'>ab191433</a>) at 1/5000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast cell line) 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: 120 kDa,31 kDa

Observed band size: 150 kDa,50 kDa

true

Exposure time: 30s