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.

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 37kDa.
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 ab260996 (JMJD6 knockout cell lysate ab257490). 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.

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 HEK293 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 line ab266402 (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.

Homozygous: 1 bp deletion in exon 1