RRAS Antibody

What are the key specifications for commercially available RRAS antibodies?

RRAS antibodies are available with varying specifications depending on research needs. The most common RRAS antibody specifications include reactivity with human and monkey samples, with a typical molecular weight detection of approximately 24 kDa . Commercially available antibodies are commonly derived from rabbit sources and demonstrate endogenous sensitivity . When selecting an RRAS antibody, researchers should consider species cross-reactivity, which is typically indicated in product documentation with key indicators such as H (Human) and Mk (Monkey) to ensure compatibility with experimental models .

What are the recommended applications and dilutions for RRAS antibodies?

RRAS antibodies are most commonly used for Western Blotting (WB) and Immunoprecipitation (IP) techniques . For optimal results in Western Blotting applications, a dilution of 1:1000 is generally recommended, while Immunoprecipitation protocols typically require a more concentrated preparation at 1:50 dilution . These dilution ratios are established to balance sensitivity and specificity in detecting endogenous RRAS protein. Researchers should validate these dilutions in their specific experimental systems, as optimal concentrations may vary depending on sample type and detection methods.

How do researchers distinguish between RRAS and other RAS family members in experimental systems?

Distinguishing RRAS from other RAS family members requires careful antibody selection and experimental design. Researchers can employ several strategies to ensure specificity:

• Select antibodies targeting the N-terminal region of RRAS, which differs significantly from other RAS proteins. For instance, some laboratories have generated rabbit polyclonal antisera against the N-terminal 23 amino acids of R-Ras specifically for this purpose .

• Include appropriate controls in experiments, such as RRAS knockout samples or cells with RRAS knockdown, to validate antibody specificity.

• Use complementary detection methods such as mass spectrometry to confirm antibody specificity when first establishing experimental protocols.

• Consider the molecular weight differences - RRAS typically appears at approximately 24 kDa, which can help distinguish it from other RAS family members on Western blots .

How can RRAS antibodies be utilized to study RAS-dependent oncogenic signaling?

RRAS antibodies serve as critical tools for investigating RAS-dependent oncogenic signaling in cancer research. Advanced studies have employed antibodies that specifically recognize activated GTP-bound RAS to develop therapeutic approaches for RAS-driven cancers . These antibodies can competitively bind to the conformationally variant regions of RAS where signaling effector molecules typically interact, thereby inhibiting downstream oncogenic signaling .

Methodologically, researchers can employ RRAS antibodies to:

• Evaluate RAS activation states through pull-down assays that distinguish GTP-bound (active) from GDP-bound (inactive) RRAS.

• Assess interactions between RRAS and downstream effectors like PI3K, RAF, and RALGDS through co-immunoprecipitation studies.

• Monitor therapeutic efficacy of RAS-targeting approaches in preclinical models, such as in the case of single domain intrabodies that have shown promise in preventing RAS-dependent tumorigenesis in mouse models .

These applications have demonstrated that antibody-based targeting of activated RAS can effectively rescue the untransformed phenotype in cancer cells harboring RAS mutations, highlighting the therapeutic potential of this approach .

What role do RRAS antibodies play in studying immune cell functions?

RRAS antibodies have been instrumental in uncovering the previously unrecognized functions of RRAS in immune regulation, particularly in dendritic cells (DCs) and T-cell responses. Studies using RRAS knockout mouse models have revealed that RRAS is expressed in multiple murine immune cells and plays critical roles in DC maturation and T-cell priming .

For researchers investigating immune cell functions, RRAS antibodies enable:

• Detection of RRAS expression across different immune cell populations through flow cytometry or immunoblotting.

• Assessment of RRAS-dependent signaling pathways downstream of Toll-like Receptor 4 (TLR4) activation in DCs.

• Investigation of immunological synapse formation between DCs and T cells, which has been shown to be impaired in RRAS-deficient models .

Methodologically, researchers typically combine RRAS antibodies with other immune markers such as PE-Cy7–CD11c, PE-CD86, FITC-MHC class II, and APC–CD40 to characterize DC populations and their maturation status in relation to RRAS expression .

How can researchers use RRAS antibodies to investigate RASopathy-associated mutations?

Constitutional dysregulation of RRAS function has been associated with RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits, and predisposition to certain malignancies . Researchers investigating these conditions utilize RRAS antibodies to characterize how specific RRAS mutations (e.g., p.Gly39dup and p.Val55Met) affect protein function and signaling.

Methodological approaches include:

• Comparing wild-type and mutant RRAS binding behaviors with effector proteins such as PIK3CA, RAF1, PLCE1, RASSF5, and RALGDS through co-immunoprecipitation assays.

• Assessing the activation state of RRAS proteins through pull-down assays that reveal the proportion of active, GTP-bound forms.

• Measuring downstream signaling through the MAPK and PI3K/AKT cascades using phospho-specific antibodies targeting key pathway components .

These studies have demonstrated that RASopathy-causing RRAS mutations are activating and promote signaling perturbation by enhancing stimulus-dependent MEK, ERK, and to a lesser extent, AKT phosphorylation .

How do RRAS antibodies contribute to understanding endothelial cell regulation?

RRAS antibodies have provided significant insights into the role of RRAS in regulating endothelial cell (EC) function and vascular development. Research has revealed that RRAS activation in ECs enforces quiescence through specific signaling pathways, with important implications for angiogenesis and vascular stabilization .

Experimental approaches using RRAS antibodies in vascular biology include:

• Tracking RRAS-mediated regulation of endothelial Jagged1 in vivo using mouse models with EC-specific Rras gene ablation (cdh5-Cre; Rras) .

• Assessing the impact of RRAS on EC-EC interactions through VE-cadherin stabilization at adherens junctions.

• Investigating RRAS-mediated activation of Akt1 and Akt2, which promotes lumen formation in sprouting blood vessels through stabilization of microtubule cytoskeleton .

• Evaluating EC proliferation in RRAS knockout models through Ki-67 immunostaining, which has revealed increased EC proliferation in Rras-deficient retinas compared to controls .

These applications have demonstrated that RRAS plays crucial roles in vascular development and homeostasis, making RRAS antibodies essential tools for researchers in the field of vascular biology.

What methodological considerations are important when using RRAS antibodies to study Notch signaling in endothelial cells?

RRAS has been shown to impact Notch signaling in endothelial cells, affecting the expression of downstream targets like Hey1, Hes1, p21, and p53 . When using RRAS antibodies to investigate these pathways, researchers should consider several methodological approaches:

• Combine RRAS antibodies with those targeting Notch pathway components to assess co-localization and potential direct interactions.

• Implement complementary techniques such as RT-qPCR to distinguish between transcriptional and post-transcriptional regulation. For instance, R-Ras38V expression increases Hey1 and p21 mRNA levels, while p53 appears to be regulated post-transcriptionally or through protein stability mechanisms .

• Incorporate γ-secretase inhibitors like N-[N-(3,5-difluorophenacetyl)-L-alanyl] to determine whether observed effects are dependent on Notch activation.

• Use in vitro co-culture methods to investigate cell-cell interactions, such as the demonstrated ability of RRAS-expressing ECs to arrest the cell cycle of adjacent cells through Jagged1-dependent mechanisms .

These methodological considerations enable researchers to delineate the complex relationships between RRAS, Notch signaling, and endothelial cell behavior in both developmental and pathological contexts.

What are common challenges in RRAS antibody-based experiments and how can they be addressed?

When working with RRAS antibodies, researchers may encounter several technical challenges that can affect experimental outcomes:

• Cross-reactivity with other RAS family members: Due to sequence homology between RAS proteins, antibodies may cross-react with multiple family members. To address this issue, researchers should:

  • Perform validation experiments using knockout or knockdown controls

  • Select antibodies targeting unique regions of RRAS, such as the N-terminal domain

  • Include appropriate positive and negative controls in all experiments

• Detection of specific activation states: Distinguishing between GTP-bound (active) and GDP-bound (inactive) RRAS requires specialized approaches:

  • Use pull-down assays with effector domain-containing proteins that preferentially bind active RRAS

  • Implement proper positive controls such as cells expressing constitutively active RRAS mutants

  • Ensure rapid sample processing to preserve activation states, as GTP hydrolysis can occur during lengthy protocols

• Variability in immunofluorescence staining: When using RRAS antibodies for localization studies, researchers may encounter:

  • High background signal, which can be reduced through optimization of blocking conditions and antibody concentrations

  • Fixation-dependent artifacts, addressed by comparing multiple fixation methods

  • Cell-type specific variability, requiring protocol adjustments based on the experimental system

How can researchers validate the specificity of RRAS antibodies in their experimental systems?

Antibody validation is crucial for ensuring reliable and reproducible results. For RRAS antibodies, a comprehensive validation approach should include:

• Genetic controls:

  • Use RRAS knockout or knockdown cell lines/tissues as negative controls

  • Include overexpression systems with tagged RRAS variants for positive controls

  • Compare results between multiple cell types or tissues with varying RRAS expression levels

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide

  • Compare results from multiple antibodies targeting different epitopes of RRAS

  • Confirm observed molecular weight matches the expected size for RRAS (approximately 24 kDa)

• Functional validation:

  • Correlate antibody detection with known functional outcomes of RRAS activation or inhibition

  • Verify expected changes in RRAS detection following stimuli known to activate RRAS signaling

  • Compare results across multiple experimental techniques (Western blotting, immunoprecipitation, immunofluorescence)

How are RRAS antibodies being used to develop therapeutic strategies for RAS-driven cancers?

The development of antibodies that specifically target activated RAS represents a promising therapeutic approach for RAS-driven cancers, which have historically been considered "undruggable" with small molecules . Current research directions include:

• Single domain antibodies (iDabs) as intrabodies: Research has demonstrated that single immunoglobulin variable region domains can specifically bind to activated GTP-bound RAS and prevent RAS-dependent tumorigenesis in mouse models . These intrabodies competitively bind to the conformationally variant regions of RAS where signaling effector molecules interact, effectively inhibiting oncogenic signaling .

• Subcellular targeting strategies: Studies have shown that plasma membrane-targeted single domain intrabodies can effectively inhibit signaling by mutant RAS, highlighting the importance of proper subcellular localization for therapeutic efficacy . Experimental approaches have employed retroviral vectors expressing FLAG-tagged intrabodies with different subcellular localization signal peptides to determine optimal targeting strategies .

• In vivo efficacy assessment: The therapeutic potential of anti-RAS intrabodies has been evaluated through tumor formation assays in athymic nude mice. For example, mice injected with cancer cells expressing plasma membrane-targeted anti-RAS intrabodies showed significant inhibition of tumor formation compared to controls .

These approaches represent a promising direction for developing macromolecular therapeutics that can directly interfere with oncogenic RAS function in human cancers.

What novel insights have RRAS antibodies provided about RASopathies and related developmental disorders?

RRAS antibodies have been instrumental in identifying and characterizing the role of RRAS mutations in RASopathies, providing important insights into these developmental disorders:

• Identification of germline mutations: Research using RRAS antibodies has helped identify specific germline mutations (p.Gly39dup and p.Val55Met) in RRAS that are associated with phenotypes within the RASopathy spectrum .

• Characterization of mutation effects: Functional studies have demonstrated that these RASopathy-causing RRAS mutations are activating and promote signaling perturbation by enhancing stimulus-dependent MEK, ERK, and to a lesser extent, AKT phosphorylation .

• Protein interaction networks: RRAS antibodies have enabled the characterization of aberrant binding behaviors of RRAS mutants with effector proteins. For example, RRAS G39dup exhibits increased binding affinity towards PIK3CA, RAF1, PLCE1, and RASSF5, while RRAS V55M shows increased affinity for RALGDS .

• Connection to cancer predisposition: The same gene that harbors germline mutations causing developmental disorders has been found to acquire somatic mutations in aggressive forms of juvenile myelomonocytic leukemia, representing an archetypal somatic RASopathy that can rapidly progress to acute myeloid leukemia .

These findings highlight the dual role of RRAS in both developmental disorders and cancer, making RRAS antibodies valuable tools for studying the molecular basis of these conditions.