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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-RhoA antibody [EPR18134] (ab187027)

Reviews (1) Submit a question References (62)
Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (ab187027)
  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Flow Cytometry (Intracellular) - Anti-RhoA antibody [EPR18134] (ab187027)
  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
  • Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (ab187027)
  • Anti-RhoA antibody [EPR18134] (ab187027)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18134] to RhoA
  • Suitable for: Flow Cyt (Intra), WB, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free PE

Overview

  • Product name

    Anti-RhoA antibody [EPR18134]
    See all RhoA primary antibodies

  • Description

    Rabbit monoclonal [EPR18134] to RhoA

  • Host species

    Rabbit

  • Tested applications

    Suitable for: Flow Cyt (Intra), WB, ICC/IFmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Human RhoA full length protein; HeLa, HEK-293 C6, Raw264.7 and NIH/3T3 cell lysates, human fetal brain and fetal kidney lysates, mouse brain, kidney and spleen lysates, rat brain, kidney and spleen lysates. ICC/IF: Jurkat and K562 cells. Flow Cyt (intra): HeLa cells.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab187027 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
1/200.
WB (1)
1/5000. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa).
ICC/IF
1/150.
Notes
Flow Cyt (Intra)
1/200.
WB
1/5000. Detects a band of approximately 22 kDa (predicted molecular weight: 22 kDa).
ICC/IF
1/150.

Target

  • Function

    Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders. May be an activator of PLCE1. Activated by ARHGEF2, which promotes the exchange of GDP for GTP.

  • Sequence similarities

    Belongs to the small GTPase superfamily. Rho family.

  • Domain

    The basic-rich region is essential for yopT recognition and cleavage.

  • Post-translational
    modifications

    Substrate for botulinum ADP-ribosyltransferase.
    Cleaved by yopT protease when the cell is infected by some Yersinia pathogens. This removes the lipid attachment, and leads to its displacement from plasma membrane and to subsequent cytoskeleton cleavage.
    AMPylation at Tyr-34 and Thr-37 are mediated by bacterial enzymes in case of infection by H.somnus and V.parahaemolyticus, respectively. AMPylation occurs in the effector region and leads to inactivation of the GTPase activity by preventing the interaction with downstream effectors, thereby inhibiting actin assembly in infected cells. It is unclear whether some human enzyme mediates AMPylation; FICD has such ability in vitro but additional experiments remain to be done to confirm results in vivo.
    Ubiquitinated by the BCR(BACURD1) and BCR(BACURD2) E3 ubiquitin ligase complexes, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration.

  • Cellular localization

    Cell membrane. Cytoplasm > cytoskeleton.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Aplysia ras related homolog 12 antibody
    • ARH12 antibody
    • ARHA antibody
    • H 12 antibody
    • H12 antibody
    • Oncogene RHO H12 antibody
    • Ras homolog family member A antibody
    • Ras homolog gene family member A antibody
    • Rho A antibody
    • Rho cDNA clone 12 antibody
    • RHO H12 antibody
    • RHO12 antibody
    • RHOA antibody
    • RHOA_HUMAN antibody
    • RHOH12 antibody
    • Small GTP binding protein Rho A antibody
    • Transforming protein Rho A antibody
    • Transforming protein RhoA antibody
    see all

Images

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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 Cytometry (Intracellular) - Anti-RhoA antibody [EPR18134] (ab187027)
    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. 

     

     

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    Western blot - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody [EPR18134] (ab187027)
    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.

  • Anti-RhoA antibody [EPR18134] (ab187027)
    Anti-RhoA antibody [EPR18134] (ab187027)

References (62)

Publishing research using ab187027? Please let us know so that we can cite the reference in this datasheet.

ab187027 has been referenced in 62 publications.

  • Xie J  et al. Energy expenditure during cell spreading influences the cellular response to matrix stiffness. Biomaterials 267:120494 (2021). PubMed: 33130323
  • He L  et al. Actin-granule formation is an additional step in cardiac myofibroblast differentiation. Ann Transl Med 9:165 (2021). PubMed: 33569467
  • Ke W  et al. The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells. Cell Prolif 54:e12987 (2021). PubMed: 33415745
  • Wang K  et al. Vertebral-specific activation of the CX3CL1/ICAM-1 signaling network mediates non-small-cell lung cancer spinal metastasis by engaging tumor cell-vertebral bone marrow endothelial cell interactions. Theranostics 11:4770-4789 (2021). PubMed: 33754027
  • Duan Z  et al. Circ_0074027 contributes to non-small cell lung cancer progression through positively modulating RHOA via sequestering miR-2467-3p. J Bioenerg Biomembr 53:223-233 (2021). PubMed: 33687619
View all Publications for this product

Customer reviews and Q&As

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Western blot abreview for Anti-RhoA antibody [EPR18134]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Human Cancer Cell line)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
10 µg
Specification
Human Cancer Cell line
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Submitted Nov 12 2021

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