Anti-RHOG antibody [1F3-RHOG]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RHOG antibody [1F3-RHOG] (AB254147)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat cells labelling RHOG with ab254147 at 1/50 dilution, followed by ab150113 goat anti-mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Jurkat cells. ab179513 anti-beta Tubulin rabbit monoclonal antibody at a 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/500 dilution was used to counterstain tubulin (Red). The nuclear counterstain was DAPI (Blue).

Negative control 1 : ab254147 at a 1/50 dilution followed by ab150080 at a 1/500 dilution.

Negative control 2 : ab179513 at a 1/200 dilution followed by ab150113 at a 1/1000 dilution.

• IP

Unknown

Immunoprecipitation - Anti-RHOG antibody [1F3-RHOG] (AB254147)

RHOG was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte), whole cell lysate with ab254147 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254147 at 1/1000 dilution. Mouse IgG for IP (HRP) (ab131368) was used at 1/1000 dilution.

Lane 1 : Jurkat whole cell lysate 10μg.

Lane 2 : ab254147 IP in Jurkat whole cell lysate.

Lane 3 : Mouse monoclonal IgG2b instead of ab254147 in Jurkat whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-RHOG antibody [1F3-RHOG] (ab254147)

Predicted band size: 21 kDa

Observed band size: 18 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RHOG antibody [1F3-RHOG] (AB254147)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized J774A.1 cells labelling RHOG with ab254147 at 1/50 dilution, followed by ab150113 goat anti-mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in J774A.1 cells. ab179513 anti-beta Tubulin rabbit monoclonal antibody at a 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/500 dilution was used to counterstain tubulin (Red). The nuclear counterstain was DAPI (Blue).

Negative control 1 : ab254147 at a 1/50 dilution followed by ab150080 at a 1/500 dilution.

Negative control 2 : ab179513 at a 1/200 dilution followed by ab150113 at a 1/1000 dilution.

• IP

Unknown

Immunoprecipitation - Anti-RHOG antibody [1F3-RHOG] (AB254147)

RHOG was immunoprecipitated from 0.35 mg J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate with ab254147 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254147 at 1/1000 dilution. Mouse IgG for IP (HRP) (ab131368) was used at 1/1000 dilution.

Lane 1 : J774A.1 whole cell lysate 10μg.

Lane 2 : ab254147 IP in J774A.1 whole cell lysate.

Lane 3 : Mouse monoclonal IgG2b instead of ab254147 in J774A.1 whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-RHOG antibody [1F3-RHOG] (ab254147)

Predicted band size: 21 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-RHOG antibody [1F3-RHOG] (AB254147)

False colour image of Western blot : Anti-RHOG antibody [1F3-RHOG] staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab254147 was shown to bind specifically to RHOG. A band was observed at 18 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in RHOG knockout cell line ab266753 (knockout cell lysate ab263328). To generate this image, wild-type and RHOG knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-RHOG antibody [1F3-RHOG] (ab254147) at 1/1000 dilution

Lane 1:

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

Lane 2:

RHOG knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human RHOG knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-rhog-knockout-hek-293t-cell-line-ab266753'>ab266753</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 21 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-RHOG antibody [1F3-RHOG] (AB254147)

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

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 18505794).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-RHOG antibody [1F3-RHOG] (ab254147) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 2:

MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 4:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate at 20 µg

Lane 5:

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

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 21 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-RHOG antibody [1F3-RHOG] (AB254147)

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

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-RHOG antibody [1F3-RHOG] (ab254147) at 1/1000 dilution

Lane 1:

Mouse bone marrow tissue lysate at 20 µg

Lane 2:

Rat thymus tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 21 kDa

Observed band size: 18 kDa

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