All lanes : Anti-OTUB1 antibody [EPR13028(B)] (ab175200) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : OTUB1 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 31 kDa

Lanes 1-2: Merged signal (red and green). Green - ab175200 observed at 31 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab175200 Anti-OTUB1 antibody [EPR13028(B)] was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°CC at 1 in 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.

 

All lanes : Anti-OTUB1 antibody (ab101471) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : OTUB1 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab101471 observed at 130 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab101471 Anti-OTUB1 antibody was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 ug/ml 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.

 

 

Allele-1: 19 bp deletion in exon 1

 

Allele-2: Insertion of the selection cassette in exon 1.

 

Representative images of OTUB1 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.