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

All lanes : Anti-RHOG antibody [1F3-RHOG] (ab254147) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : RHOG knockout HEK-293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

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

Lane 1 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 4 : J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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.

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.

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

Lane 1 : Mouse bone marrow tissue lysate
Lane 2 : Rat thymus tissue lysate

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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

Exposure time: 3 minutes.

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.

RHOG was immunoprecipitated from 0.35 mg J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate with abab254147 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.