Anti-KDM6A / UTX antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-KDM6A / UTX antibody (AB36938)

ICC/IF image of ab36938 stained MCF7 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab36938, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa cells fixed in 4% PFA at 1ug/ml and HeLa and MCF7 100% methanol fixed cells at 1ug/ml. However, this Fast-Track antibody is not yet fully characterised. This image represents inconclusive preliminary data.

• WB

Lab

Western blot - Anti-KDM6A / UTX antibody (AB36938)

Lanes 1- 2 : Merged signal (red and green). Green - ab36938 observed at 154 -157 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab36938 was shown to react with KDM6A / UTX in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265110 (knockout cell lysate ab257214) was used. Wild-type HeLa and KDM6A knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab36938 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 Dilution and a 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.

All lanes:

Western blot - Anti-KDM6A / UTX antibody (ab36938) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

KDM6A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human KDM6A (UTX) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-kdm6a-utx-knockout-hela-cell-line-ab265110'>ab265110</a>)

Predicted band size: 154 kDa

Observed band size: 154-157 kDa

false

• WB

Lab

Western blot - Anti-KDM6A / UTX antibody (AB36938)

Lanes 1 - 2 : Green - ab36938 observed at 154 kDa.

Lanes 3 - 4 : Red - loading control, ab18058, observed at 130 kDa.

ab36938 was shown to recognize KDM6A in wild-type HAP1 cells as signal was lost at the expected MW in KDM6A knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and KDM6A knockout samples were subjected to SDS-PAGE. ab36938 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 0.7 μg/ml and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-KDM6A / UTX antibody (ab36938) at 0.7 µg/mL

Lanes 3 - 4:

Anti-Vinculin antibody [SPM227] (<a href='/en-us/products/unavailable/vinculin-antibody-spm227-ab18058'>ab18058</a>) at 1/20000 dilution

Lanes 1 and 3:

Wild-type HAP1 whole cell lysate at 20 µg

Lanes 2 and 4:

KDM6A knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 154 kDa

false

• WB

Project

Western blot - Anti-KDM6A / UTX antibody (AB36938)

This Fast-Track antibody is not yet fully characterised. This image represents inconclusive preliminary data.

All lanes:

Western blot - Anti-KDM6A / UTX antibody (ab36938) at 1 µg/mL

Lane 1:

Testis (Rat) Tissue Lysate - normal tissue (<a href='/en-us/products/unavailable/testis-rat-tissue-lysate-normal-tissue-ab29388'>ab29388</a>) at 10 µg

Lane 2:

Testis (Mouse) Tissue Lysate at 10 µg

Lane 3:

Liver (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG -H&L- Pre Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 154 kDa

Observed band size: 154 kDa,157 kDa,80 kDa

false

• WB

AbReview8991****

Western blot - Anti-KDM6A / UTX antibody (AB36938)

Lane 3 is NIH3T3 cells transduced with lentivirus expressing shRNA for mouse Utx.

5% milk was used as the blocking agent (incubated for 1 hour at 25°C).

ab36938 was diluted with TBS+0.1% Tween 20 and incubated for 1 hour at 25°C.

All lanes:

Western blot - Anti-KDM6A / UTX antibody (ab36938) at 1 µg/mL

Lane 1:

Whole NIH3T3 cell lysate (no shRNA) at 20 µg

Lane 2:

Whole NIH3T3 cell lysate (Control shRNA) at 20 µg

Lane 3:

Whole NIH3T3 cell lysate (Utx shRNA) at 20 µg

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit polyclonal

Predicted band size: 154 kDa

Observed band size: 148 kDa

true

Exposure time: 10s

This image is courtesy of an anonymous Abreview