Anti-KDM6A / UTX antibody [EPR23203-211]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-KDM6A / UTX antibody [EPR23203-211] (AB253183)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte), (Left panel) / HAP1 (Human chronic myelogenous leukemia near-haploid cell line), (Right panel) cells labelling KDM6A / UTX with ab253183 at 1/50 dilution (1μg) (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).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : THP-1.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-KDM6A / UTX antibody [EPR23203-211] (AB253183)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized F9 (Mouse embryonal carcinoma epithelial cell) cells labelling KDM6A / UTX with ab253183 at 1/500 dilution (0.1μg) (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).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (AB253183)

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

Fresh lysates were used in this WB.

All lanes:

Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 154 kDa

Observed band size: 160 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (AB253183)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-4 : Merged signal (red and green). Green - ab253183 observed at 160 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

ab253183 Anti-KDM6A antibody [EPR23203-211] was shown to react with KDM6A in HAP1 wild-type cells in Western blot. Loss of signal was observed when KDM6A knockout sample was used. Wild-type and KDM6A knockout samples were subjected to SDS-PAGE.

ab253183 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1/1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Negative control : THP-1 (PMID : 19330029).

The expression profile and molecular weight observed is consistent with what has been described in the literature (PMID : 19330029).

The the bands between 50kDa and 150kDa are caused by degradation based on the result of fresh lysates.

All lanes:

Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

KDM6A knockout HAP1 whole cell lysate at 20 µg

Lane 3:

F9 (mouse embryonal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 154 kDa

Observed band size: 160 kDa

false

• WB

Lab

Western blot - Anti-KDM6A / UTX antibody [EPR23203-211] (AB253183)

Western blot : Anti-KDM6A antibody [EPR23203-211] (ab253183) 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, ab253183 was shown to bind specifically to KDM6A. A band was observed at 150-160 kDa in wild-type A549 cell lysates with no signal observed at this size in KDM6A knockout cell line. To generate this image, wild-type and KDM6A 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-KDM6A / UTX antibody [EPR23203-211] (ab253183) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 7 µg

Lane 2:

KDM6A knockout A549 cell lysate at 7 µg

Lane 3:

Wild-type HAP1 cell lysate at 7 µg

Lane 4:

KDM6A knockout HAP1 cell lysate at 7 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: 154 kDa

Observed band size: 150 kDa,160 kDa

false