rhoaa Antibody

What is RhoA and why is it significant in molecular research?

RhoA is one of the most extensively studied members of the Rho GTPase family, involved in diverse cellular processes including cytoskeletal reorganization, transcription regulation, cell migration, and metastasis. As the predominant Rho isoform expressed across numerous cell types, it functions as a molecular switch that cycles between active (GTP-bound) and inactive (GDP-bound) states .

Key research significance:

• Regulates actomyosin contractility, cytokinesis, focal adhesion assembly, and cell polarity

• Mediates cell shape determination through actin polymerization mechanisms

• Implicated in multiple pathological conditions including neuroinflammation and cancer progression

How do I select the appropriate RhoA antibody for my experimental needs?

Selection depends primarily on your experimental application and specificity requirements:

When selecting, consider:

• Species cross-reactivity requirements (human, mouse, rat compatibility)

• Isoform specificity - some antibodies recognize only RhoA while others cross-react with RhoB/RhoC

• Experimental readout needs - total RhoA vs. active RhoA detection

How can I validate the specificity of my RhoA antibody?

Antibody validation requires multiple complementary approaches:

• Western blot analysis with recombinant proteins: Compare binding to purified RhoA versus related proteins (RhoB, RhoC, Rac1, Cdc42) to confirm specificity

• Knockout/knockdown controls: Utilize RhoA-deficient cells as critical negative controls

• Multiple antibody comparison: Test with at least two antibodies targeting different epitopes to confirm consistent results

• Cross-species testing: Verify consistent detection in expected molecular weight ranges across relevant species samples

Some RhoA antibodies demonstrate excellent specificity. For example, ARH05 monoclonal antibody recognizes only RhoA, not RhoB, RhoC, Rac1, Rac2, Rac3, Cdc42, or H-Ras, while other commercial antibodies may recognize one or more of these isoforms .

How can I specifically detect active (GTP-bound) RhoA in cell and tissue samples?

Active RhoA detection requires specialized techniques that preserve the GTP-bound state:

• Pull-down assays: Use GST-RBD (Rho-binding domain) fusion proteins to selectively capture GTP-bound RhoA

  • Protocol parameters: 30 μg GST-RBD with Glutathione-Sepharose 4B beads, followed by SDS-PAGE on 12.5% acrylamide gels

  • Verification: Immunoblot with anti-RhoA antibody, with parallel total RhoA immunoblotting for normalization

• GTP-RhoA specific antibodies: Use antibodies specifically recognizing the active conformation

  • Applications: Immunofluorescence imaging and immunohistochemistry to visualize active RhoA localization

  • Controls: Include GDP-loaded RhoA samples as negative controls

• FRET-based biosensors: For live-cell imaging of RhoA activation dynamics

  • Raichu-RhoA biosensor allows precise monitoring of RhoA activation/inactivation cycle in real time

  • Detected significant reduction in RhoA activity within 10 minutes of LPS exposure in microglia

What are the technical considerations for using RhoA antibodies in neuroinflammation research?

Neuroinflammation studies require special attention to:

• Activity state-specific antibodies: Use paired approaches to measure both:

  • pRhoA antibody (against S188 phosphorylation) to detect inactive RhoA

  • pLIMK antibody (against T508 phosphorylation) as a RhoA signaling indicator

• Regional analysis considerations: RhoA expression and activity show rostral-caudal differences in the brain

  • Differential expression patterns between rostral and caudal planes of hippocampus and cortex

  • Dynamic redistribution between nucleus and cytoplasm in hippocampal subregions

• Disease state comparison: RhoA activity changes in Alzheimer's disease brain tissues

  • Human AD cortex shows altered nuclear RhoA staining patterns compared to non-disease controls

  • Cerebellum exhibits striking differences in pRhoA immunoreactivity between AD and ND human brain samples

• Experimental readouts: When studying neuroinflammation processes

  • LPS treatment decreases RhoA activity (measured by FRET) within 10 minutes (IC₅₀ = 110 ng/ml)

  • Systemic LPS administration (4 mg/kg) significantly decreases GTP-RhoA in Iba-1+ microglia in vivo

What are common pitfalls when using RhoA antibodies, and how can they be addressed?

Common challenges and solutions include:

• Isoform cross-reactivity issues:

  • Problem: Many commercial antibodies cross-react with related Rho proteins

  • Solution: Use highly selective antibodies like ARH05 (recognizes only RhoA) or validate specificity using recombinant proteins

• Activity state preservation:

  • Problem: GTP-bound active state can be lost during sample preparation

  • Solution: Use rapid lysis procedures with Mg²⁺ supplementation and avoid freeze-thaw cycles

• Nuclear versus cytoplasmic localization:

  • Problem: RhoA shows differential localization patterns that change during disease states

  • Solution: Perform careful subcellular fractionation or use high-resolution imaging with nuclear counterstains

• Assay-specific limitations:

  • SDS denatures GTP-bound RhoA, making certain antibodies unsuitable for Western blot detection of active RhoA

  • For active RhoA, use immunoprecipitation, immunohistochemistry, or immunofluorescence methods instead

How do I optimize immunohistochemistry protocols for RhoA detection in tissue sections?

Optimize your IHC protocol with these research-validated parameters:

• Antibody selection: Choose antibodies validated for IHC applications

  • For total RhoA: RhoA (67B9) Rabbit mAb at 1:800 dilution

  • For active RhoA: Anti-RhoA-GTP at 1:50-1:250 dilution

• Signal amplification: For low expression tissues, consider:

  • Tyramide signal amplification (TSA) system

  • Protocol: Primary antibody incubation followed by biotinylated secondary (1:500), streptavidin HRP (1:2000), biotinylated tyramide (10 min), and streptavidin FITC (1:1000)

• Scoring system implementation: Adopt a standardized quantification system

  • Use a 3-point scale (1+, 2+, 3+) for nuclear and cytoplasmic staining

  • Include both staining intensity and percentage of positive cells in analysis

• Controls: Include critical controls for reliable interpretation:

  • Tissue from RhoA knockout/knockdown models as negative controls

  • Known positive tissues (e.g., human platelet extracts)

  • Adjacent sections with non-immune IgG at matching concentrations

How can RhoA antibodies be used to investigate disease mechanisms in cancer and neurodegeneration?

RhoA antibodies enable mechanistic investigation through multiple approaches:

Cancer research applications:

• Tumor heterogeneity assessment: RhoA expression correlates with infiltrative vessel co-optive growth patterns in hepatocellular carcinoma

• Therapeutic target identification: Treg cell-specific RhoA deletion inhibits tumor growth by promoting tumor-infiltrating effector T cells

• Metastasis mechanism investigation: Heterozygous RhoA deletion in Treg cells impacts tumor immunity without causing systemic autoimmunity

Neurodegenerative disease applications:

• Alzheimer's disease (AD) signaling analysis:

  • Rostral-caudal expression patterns of RhoA signaling components in AD brain tissue

  • Differential pRhoA expression between AD cortex and cerebellum

• Neuroinflammatory pathway assessment:

  • RhoA activity decreases within minutes of inflammatory stimulus in microglia

  • Constitutively active RhoA prevents LPS-induced ROS production and NF-κB activation

What are the latest approaches for studying RhoA-mediated signaling pathways with antibody-based methods?

Cutting-edge approaches include:

• Intracellular nanobodies:

  • Functional intracellular nanobody RH28 specifically blocks RHOA/ROCK signaling

  • Detected using tripartite split-GFP protein-protein interaction reporter system

  • Triggers elongated cellular phenotype in metastatic melanoma cells (WM266-4)

• Multi-parameter signaling assessment:

  • Combined analysis of upstream regulators and downstream effectors:

    • ROS production via FRET biosensor HSP

    • Src activation via FRET biosensor and phospho-specific antibodies

    • NF-κB activation via nuclear p65 translocation analysis

• Graded expression analysis:

  • RhoA expression levels in Treg cells distinguish tumor immunity from autoimmunity

  • Homozygous vs. heterozygous RhoA deletion reveals differential phenotypes:

    • Homozygous: fatal systemic inflammatory disorders

    • Heterozygous: maintained Treg homeostasis with increased plasticity

• FRET-based live imaging:

  • Raichu-RhoA biosensor allows real-time monitoring of activation dynamics

  • Can measure RhoA inhibition kinetics (detects changes within 10 minutes of stimulus)

What considerations should be made when designing RhoA antibody-based experiments for primary cell cultures?

Primary cell culture experiments require specialized considerations:

• Cell type-specific baseline activity:

  • Microglia exhibit rapid RhoA activity changes in response to inflammatory stimuli

  • Primary cortical microglia showed significant RhoA activity reduction within 10 minutes of LPS exposure

• Species-specific antibody validation:

  • Verify antibody reactivity with the species of your primary cells

  • Example: ARH05 is validated for human, mouse, and rat samples

• Functional readouts across cell types:

  • Cytoskeletal assessment: Different cell types show distinct morphological responses

  • Cytokine secretion: Primary microglial cultures with RhoA Q63L showed prevented secretion of TNF-α and IL-1β after LPS stimulation

• Quantification methods:

  • Use multiple complementary techniques:

    • FRET-based activity monitoring for live cells

    • Pull-down assays for biochemical quantification

    • Immunofluorescence for subcellular localization

  • Always include positive controls (e.g., constitutively active RhoA Q63L)