All lanes : Anti-KDM6A / UTX antibody (ab36938) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : KDM6A knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 154 kDa
Observed band size: 154 -157 kDa why is the actual band size different from the predicted?

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.

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.

Lanes 1-2 : Anti-KDM6A / UTX antibody (ab36938) at 0.7 µg/ml
Lanes 3-4 : Anti-Vinculin antibody [SPM227] (ab18058) at 1/20000 dilution

Lanes 1 & 3 : Wild-type HAP1 whole cell lysate
Lanes 2 & 4 : KDM6A knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 154 kDa

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.

All lanes : Anti-KDM6A / UTX antibody (ab36938) at 1 µg/ml

Lane 1 : Whole NIH3T3 cell lysate (no shRNA)
Lane 2 : Whole NIH3T3 cell lysate (Control shRNA)
Lane 3 : Whole NIH3T3 cell lysate (Utx shRNA)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated Goat anti-Rabbit polyclonal

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 154 kDa
Observed band size: 148 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

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. 

See Abreview

All lanes : Anti-KDM6A / UTX antibody (ab36938) at 1 µg/ml

Lane 1 : Testis (Rat) Tissue Lysate - normal tissue (ab29388)
Lane 2 : Testis (Mouse) Tissue Lysate
Lane 3 : Liver (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

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

Performed under reducing conditions.

Predicted band size: 154 kDa
Observed band size: 154,157 kDa why is the actual band size different from the predicted?
Additional bands at: 80 kDa. We are unsure as to the identity of these extra bands.



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