Anti-RhoA antibody [EPR18134]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (AB187027)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (AB187027)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RhoA antibody [EPR18134] (AB187027)

Intracellular 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.

• WB

Lab

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

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:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/1000 dilution

Lane 1:

Wild-type Hek293T whole cell lysate at 20 µg

Lane 2:

RHOA knockout Hek293T whole cell lysate at 20 µg

Lane 3:

Jurkat whole cell lysate at 20 µg

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

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.

All lanes:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lanes 1 and 3:

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

Lanes 2 and 4:

RHOA knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human RHOA knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-rhoa-knockout-hek-293t-cell-line-ab266592'>ab266592</a>)

Predicted band size: 22 kDa

Observed band size: 21 kDa

false

• WB

Supplier Data

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human epithelial cells from embryonic kidney) 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 1/1000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

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.

All lanes:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1:

Human RhoA full length protein at 0.01 µg

Lane 2:

Human RhoB full length protein at 0.01 µg

Lane 3:

Human RhoC full length protein at 0.01 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 1/1000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-RhoA antibody [EPR18134] (AB187027)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-RhoA antibody [EPR18134] (ab187027) at 1/5000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Lane 5:

Rat kidney lysate at 10 µg

Lane 6:

Rat spleen lysate at 10 µg

Lane 7:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 8:

Raw264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 9:

NIH/3T3 (Mouse embryo fibroblast cells) whole cell lysate at 10 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 1/1000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 3min