All lanes : Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lanes 1 & 3 : Wild-type HEK-293T cell lysate
Lanes 2 & 4 : RHOA knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 21 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab187027 observed at 21 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab187027 was shown to react with RhoA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266592 (knockout cell lysate ab257637) was used. Wild-type HEK-293T and RHOA knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab187027 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 5000 dilution and a 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling RhoA with ab187027 at 1/150 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Jurkat cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
1. ab187027 at 1/150 dilution followed by ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

All lanes : Anti-RhoA antibody [EPR18134] (ab187027) at 1/1000 dilution

Lane 1 : Wild-type Hek293T whole cell lysate
Lane 2 : RHOA knockout Hek293T whole cell lysate
Lane 3 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 22 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab187027 observed at 22 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab187027 was shown to specifically react with RhoA in wild-type Hek293T cells as signal was lost in RHOA knockout cells. Wild-type and RHOA knockout samples were subjected to SDS-PAGE. Ab187027 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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 : Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1 : Human RhoA full length protein
Lane 2 : Human RhoB full length protein
Lane 3 : Human RhoC full length protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 1 second

5% NFDM/TBST: Blocking and diluting buffer.

Human RhoA full length protein (ab101594) contains aa1-193 with His-Tag^® at N-Terminus; Human RhoB full length protein (ab107139) contains aa1-193 with His-Tag^® at N-Terminus; Human RhoC full length protein (ab98085) contains aa1-190 with His-Tag^® at N-Terminus.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling RhoA with ab187027 at 1/200 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and a unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

All lanes : Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat kidney lysate
Lane 6 : Rat spleen lysate
Lane 7 : C6 (Rat glial tumor cells) whole cell lysate
Lane 8 : Raw264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 9 : NIH/3T3 (Mouse embryo fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling RhoA with ab187027 at 1/150 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on K562 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
1. ab187027 at 1/150 dilution followed by ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.