All lanes : Anti-JMJD6 antibody (ab64575) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : JMJD6 Knockout HEK293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab64575 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab64575 was shown to react with JMJD6 in wild-type HEK293T cells in Western blot with loss of signal observed in JMJD6 knockout cell line ab266402 (JMJD6 knockout cell lysate ab260996). Wild-type and JMJD6 knockout HEK293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab64575 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

ICC/IF image of ab64575 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64575, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% formaldehyde fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.

All lanes : Anti-JMJD6 antibody (ab64575) at 1 µg/ml

Lane 1 : THP1 cell lysate
Lane 2 : Wild-type HEK293 cell lysate
Lane 3 : JMJD6 knockout HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab64575 observed at 50 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab64575 was shown to react with JMJD6 in wild-type HEK-293 cells in western blot. Loss of signal was observed when JMJD6 knockout cell lysate (ab257490) was used. Wild-type and JMJD6 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in non-mammalian (TBS-based) blocking solution before incubation with ab64575 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-JMJD6 antibody (ab64575) at 1 µg/ml

Lane 1 : Liver (Rat) Tissue Lysate
Lane 2 : Liver (Mouse) Tissue Lysate
Lane 3 : Heart (Mouse) 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: 46 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?
Additional bands at: 26 kDa, 30 kDa, 43 kDa (possible isoform). We are unsure as to the identity of these extra bands.


Exposure time: 16 minutes