Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (AB232581)

ab175200 (purified) at 1 : 50 dilution (2ug) immunoprecipitating OTUB1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab175200 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab175200 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

All lanes:

Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (<a href='/en-us/products/primary-antibodies/otub1-antibody-epr13028b-ab175200'>ab175200</a>)

Predicted band size: 31 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (AB232581)

Western blot analysis on immunoprecipitation pellet from MCF7 cell lysate labeling OTUB1 with unpurified ab175200 at 1/10 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

All lanes:

Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (<a href='/en-us/products/primary-antibodies/otub1-antibody-epr13028b-ab175200'>ab175200</a>)

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (AB232581)

This data was developed using the same antibody clone in a different buffer formulation (ab175200).

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:

Western blot - Anti-OTUB1 antibody [EPR13028(B)] (<a href='/en-us/products/primary-antibodies/otub1-antibody-epr13028b-ab175200'>ab175200</a>) at 1/1000 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,31 kDa

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• WB

Supplier Data

Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (AB232581)

Lanes 1 - 4 : Merged signal (red and green). Green - ab175200 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab175200 was shown to specifically react with OTUB1 in wild type cells as signal was lost in OTUB1 knockout cells. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1,000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

All lanes:

Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581) at 1/1000 dilution

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:

HEK-293 whole cell lysate at 20 µg

Predicted band size: 31 kDa

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