Anti-OTUB1 antibody [EPR24917-75]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HEK293T (human embryonic kidney epithelial cell, Right)/ OTUB1 KO HEK293T (Left) cells labelling OTUB1 with ab270959 at 1/500 dilution (0.1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Positive staining on 293T cells (ab255449), while no staining on OTUB1 knockout HEK-293T cells (ab266551).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling OTUB1 with ab270959 at 1/250 (2.204 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear and cytoplasmic staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling OTUB1 with ab270959 at 1/500 dilution (0.1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized OTUB1 KO HEK293T (ab266551) cells labelling OTUB1 with ab270959 at 1/250 (2.204 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing no staining in OTUB1 KO HEK293T cell line and nuclear and cytoplasmic staining in Parental HEK293T is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

OTUB1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10ug with ab270959 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270959 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10ug

Lane 2 : ab270959 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270959 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 7 seconds

All lanes:

Immunoprecipitation - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

Predicted band size: 31 kDa

false

• WB

Lab

Western blot - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 26 seconds

All lanes:

Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

U-2 OS(human bone osteosarcoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse brain tissue lysate at 20 µg

Lane 5:

Rat liver tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 31 kDa

false

• WB

Lab

Western blot - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Lanes 1-3 : Merged signal (red and green). Green -ab270959 observed at 31kDa. Red - loading control ab8245 observed at 36 kDa. ab270959 Anti-OTUB1 antibody [EPR24917-75] was shown to specifically react with OTUB1 in wild-type HEK293T 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.

ab270959 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

OTUB1 knockout HEK293T (<a href='/en-us/products/cell-lines/human-otub1-knockout-hek-293t-cell-line-ab266551'>ab266551</a>) whole 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>)

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 31 kDa

false

• WB

Lab

Western blot - Anti-OTUB1 antibody [EPR24917-75] (AB270959)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 15 seconds

All lanes:

Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Lane 2:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 31 kDa

false