Anti-OTUB1 antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody (AB101471)

ICC/IF image of ab101471 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab101471, 5μg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, goat anti-rabbit DyLight® 488 (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 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5μg/ml, and in 100% methanol fixed (5 min)HeLa, HepG2 and MCF7 cells at 5μg/ml.

• WB

Lab

Western blot - Anti-OTUB1 antibody (AB101471)

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.

All lanes:

Western blot - Anti-OTUB1 antibody (ab101471) at 1/10000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

OTUB1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human OTUB1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-otub1-knockout-hek-293t-cell-line-ab266551'>ab266551</a>)

Predicted band size: 31 kDa

Observed band size: 130 kDa

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• WB

Lab

Western blot - Anti-OTUB1 antibody (AB101471)

Lanes 1 - 4 : Merged signal (red and green). Green - ab101471 observed at 31 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab101471 was shown to recognize OTUB1 in wild-type HAP1 cells as signal was lost at the expected MW in OTUB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1μg/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:

Western blot - Anti-OTUB1 antibody (ab101471) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

OTUB1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

Hek293 whole cell lysate at 20 µg

Predicted band size: 31 kDa

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• WB

Unknown

Western blot - Anti-OTUB1 antibody (AB101471)

Secondary antibody - goat anti-rabbit HRP (ab97080)

All lanes:

Western blot - Anti-OTUB1 antibody (ab101471) at 1 µg/mL

Lane 1:

Brain (Mouse) Tissue Lysate at 10 µg

Lane 2:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa,62 kDa

true

Exposure time: 12min