RRAS2 Antibody

What is RRAS2 and why is it significant in research?

RRAS2 (Related RAS Viral R-Ras Oncogene Homolog 2), also known as TC21, is a small GTP-binding protein belonging to the Ras superfamily of GTPases. It functions as a signal transducer located in the plasma membrane and regulates cell proliferation and differentiation via the RAS/MAPK cascade. RRAS2 has significant research importance due to its high oncogenic potential, with overexpression and mutations reported in various tumor tissues and cell lines. The RRAS2 protein shows 55-60% amino acid identity with the classical Ras subfamily (H-Ras, K-Ras, and N-Ras), which have been widely investigated in human cancer development. RRAS2 has been found to be involved in different physiological functions, including platelet activation, Schwann cell migration, and mammary gland development, making it a multifaceted research target across different biological systems .

What cellular functions does RRAS2 regulate?

RRAS2 regulates multiple cellular processes that are critical for normal development and disease pathogenesis. In the immune system, RRAS2 interacts directly with both B and T cell receptors through their immunoreceptor tyrosine-based activation motif (ITAM), predominantly in its inactive GDP-bound form, providing tonic survival signals. In T cells, RRAS2 regulates TCR internalization after immune synapse formation, but only in its wild-type form. For B cells, RRAS2 controls the correct formation of germinal centers by regulating B cell metabolism. Beyond immune functions, RRAS2 regulates platelet activation through interaction with the glycoprotein VI-ITAM-containing collagen receptor, controls Schwann cell migration in the central nervous system, and directs proper mammary gland development. These diverse roles make RRAS2 a critical research target across multiple biological systems .

What types of RRAS2 antibodies are available for research?

Various types of RRAS2 antibodies are available for research applications, differing in host species, clonality, conjugation status, and detection methods. Monoclonal antibodies include clones such as EM-50, 2D3-4B8, and 15C4, produced in mouse hosts with specific epitope recognition properties. For example, the EM-50 clone specifically recognizes R-Ras2/TC21 protein and does not cross-react with R-Ras1, H-Ras, K-Ras, or N-Ras. Polyclonal antibodies are available from rabbit hosts, offering broader epitope recognition. Several conjugated versions exist, including PE (R-phycoerythrin) conjugated antibodies optimized for flow cytometry applications. Different antibodies target specific regions of the RRAS2 protein, such as the C-terminal region, central domain, or full-length protein (AA 1-204), allowing researchers to select antibodies based on their experimental requirements and the specific epitope of interest .

What are the most common applications for RRAS2 antibodies?

RRAS2 antibodies are utilized across multiple experimental techniques in research settings. Western blotting (WB) represents a primary application, allowing researchers to detect and quantify RRAS2 protein expression in various tissue samples and cell lines. Flow cytometry (FACS) applications, particularly with PE-conjugated antibodies, enable intracellular staining and quantitative assessment of RRAS2 expression at the single-cell level. Enzyme-linked immunosorbent assay (ELISA) provides another quantitative approach for detecting RRAS2 in solution. Immunohistochemistry (IHC) and immunofluorescence (IF) techniques allow for visualization of RRAS2 expression patterns within tissue sections and cells, offering insights into localization and distribution. Additionally, RRAS2 antibodies are used in immunocytochemistry (ICC) for examining protein distribution in cultured cells. This diversity of applications makes RRAS2 antibodies versatile tools in both basic research and translational studies investigating Ras-related signaling pathways .

How can RRAS2 antibodies be used to study cancer development and progression?

RRAS2 antibodies serve as critical tools for investigating the role of RRAS2 in oncogenesis across multiple cancer types. Research has revealed RRAS2 overexpression in oral squamous cell carcinoma, esophageal tumors, hepatocellular carcinoma, and aggressive skin cancers. In experimental settings, researchers can employ Western blotting with RRAS2 antibodies to quantify expression levels in tumor versus normal tissues, establishing correlation with disease progression or patient outcomes. Immunohistochemistry applications with these antibodies allow for spatial visualization of RRAS2 distribution within tumor microenvironments, potentially revealing heterogeneity of expression. Flow cytometry with RRAS2 antibodies enables quantitative assessment of protein expression at the single-cell level in tumor samples, facilitating the identification of subpopulations with differential expression. For mechanistic studies, RRAS2 antibodies can be used in conjunction with phospho-specific antibodies targeting downstream effectors (such as phospho-ERK1/2) to evaluate RRAS2-mediated signaling activity in cancer models, as demonstrated in studies where RRAS2 variants were found to enhance ERK phosphorylation .

What methodological considerations are important when using RRAS2 antibodies for intracellular staining?

When performing intracellular staining with RRAS2 antibodies, several methodological considerations are crucial for obtaining reliable results. First, effective cell fixation and permeabilization are essential, as RRAS2 is an intracellular antigen associated with the plasma membrane. For flow cytometry applications, recommended protocols suggest using 1-4 μg/mL of antibody concentration for optimal staining results. When using PE-conjugated antibodies like clone EM-50, it's important to note that these have been purified and conjugated with R-phycoerythrin under optimum conditions, with unconjugated antibody and free fluorochrome removed by size-exclusion chromatography to minimize background signal. For validation purposes, proper controls should include isotype controls (such as IgG1 kappa for the EM-50 monoclonal antibody) to assess non-specific binding. When designing multi-parameter flow cytometry panels, consideration of fluorochrome spectral overlap is necessary, particularly when PE-conjugated antibodies are used. Additionally, researchers should be aware of potential cross-reactivity issues, though antibodies like EM-50 have been specifically developed to recognize R-Ras2/TC21 protein without cross-reactivity to related proteins like R-Ras1, H-Ras, K-Ras, and N-Ras .

How should RRAS2 antibodies be validated for specificity in experimental systems?

Comprehensive validation of RRAS2 antibodies is essential to ensure experimental reliability and reproducibility. A multi-faceted validation approach should include Western blot analysis using positive controls (tissues or cell lines known to express RRAS2) and negative controls (tissues or cells with RRAS2 knockdown). The expected molecular weight for RRAS2 protein is approximately 23-25 kDa, and validation should confirm a single band at this size. Genetic validation can be performed using RRAS2 knockout or knockdown systems, where antibody signal should be absent or significantly reduced. Competition assays, where the antibody is pre-incubated with purified RRAS2 protein before application to samples, can verify binding specificity. For monoclonal antibodies like EM-50, researchers should confirm the antibody's specificity against related Ras family proteins (R-Ras1, H-Ras, K-Ras, and N-Ras). When using RRAS2 antibodies in different species, cross-reactivity testing is necessary, as some antibodies show reactivity to human RRAS2 but not mouse or rat orthologs. For immunohistochemistry applications, antibody dilution optimization and comparison of staining patterns with published literature can provide additional validation. Implementing these rigorous validation steps ensures that experimental results accurately reflect RRAS2 biology rather than non-specific signals .

How have mouse models contributed to understanding RRAS2 function in leukemia development?

Mouse models have provided crucial insights into RRAS2's role in leukemia development, particularly chronic lymphocytic leukemia (CLL). A breakthrough model was established by increasing RRAS2 levels through genetic engineering, resulting in CLL development in 100% of cases. This model, known as Rosa26-RRAS2 fl/fl-mb1-Cre, was created by cloning the human R-RAS2 coding sequence tagged with HA into a specific vector and inserting it into the Rosa26 locus via homologous recombination. When crossed with mb1-Cre mice, which express Cre recombinase specifically in B cells from early developmental stages, this model achieves B-cell specific overexpression of RRAS2. The model has proven essential for testing potential CLL treatments before human trials, with validation studies showing that established CLL drugs such as ibrutinib and venetoclax effectively killed leukemia cells in these mice. These findings demonstrate that wild-type RRAS2 overexpression alone is sufficient to drive B-CLL development, highlighting the proto-oncogenic properties of this protein even without activating mutations. This model opens new avenues for testing novel therapeutic approaches targeting RRAS2, which has not been extensively explored in clinical settings for leukemia treatment .

What is the role of RRAS2 in Noonan syndrome and how can antibodies help study this connection?

Recent research has uncovered a significant connection between RRAS2 pathogenic variants and Noonan syndrome-like clinical features. Studies have reported patients with specific RRAS2 mutations (p.Gly23Val and p.Gly24Glu) presenting with Noonan-like phenotypes. These germline mutations differ from the somatic mutations found in various cancers but affect similar functional domains of the protein. RRAS2 antibodies play a crucial role in studying this connection through several methodological approaches. In functional studies, Western blotting with phospho-specific antibodies targeting ERK1/2 (Thr202/Tyr204) has been used to demonstrate that these pathogenic variants enhance phosphorylation of downstream MAPK pathway components, providing mechanistic insights into how RRAS2 mutations contribute to the syndrome's pathophysiology. Expression vectors containing wild-type and mutant RRAS2 have been developed for cellular studies, with antibody-based detection methods confirming proper expression. Additionally, animal models expressing human RRAS2 variants (such as HA-tagged RRAS2G23V in Drosophila) have been generated, with antibodies enabling verification of transgene expression and pathway activation. These approaches collectively demonstrate how RRAS2 antibodies facilitate the investigation of the molecular mechanisms underlying Noonan syndrome caused by RRAS2 variants .

What techniques can be used to study RRAS2 activation state in normal versus pathological conditions?

Several sophisticated techniques employing RRAS2 antibodies can be used to investigate the activation state of RRAS2 in normal versus pathological conditions. GTP-loading assays, which utilize the differential binding of active (GTP-bound) versus inactive (GDP-bound) RRAS2 to specific effector proteins, can be combined with RRAS2 antibodies for detection via Western blotting. Proximity ligation assays (PLA) can detect interactions between RRAS2 and its binding partners, providing spatial information about activation state within cells or tissues. Phospho-specific antibodies targeting downstream effectors of RRAS2 signaling, such as phospho-ERK1/2 (Thr202/Tyr204), serve as indirect indicators of RRAS2 activation. For tissue samples, dual immunohistochemistry or immunofluorescence with RRAS2 antibodies and phospho-ERK antibodies can map activation patterns across different cell types or disease stages. In the study of RRAS2 variants associated with Noonan syndrome, researchers have employed a combination of these approaches to demonstrate enhanced downstream signaling. Additionally, FRET-based biosensors for RRAS2 activation can be developed and used in conjunction with antibody validation to monitor real-time changes in RRAS2 activity in living cells under various stimulation conditions. These methodologies collectively provide a comprehensive toolkit for dissecting RRAS2 activation dynamics in both physiological and pathological contexts .

What protocol modifications are recommended when using RRAS2 antibodies for Western blotting?

When using RRAS2 antibodies for Western blotting, several protocol modifications can optimize detection and specificity. Sample preparation should include appropriate lysis buffers containing protease inhibitors to preserve RRAS2 protein integrity, with some protocols recommending additional phosphatase inhibitors when studying activation state. For gel electrophoresis, 12-15% polyacrylamide gels are preferred due to the relatively small size of RRAS2 (approximately 23-25 kDa). During transfer, using PVDF membranes rather than nitrocellulose may improve protein retention and signal strength. For blocking, 5% non-fat dry milk in TBST is commonly used, though for phospho-specific detection of downstream targets, 5% BSA may be preferred to prevent phosphatase activity in milk. Primary RRAS2 antibody incubation is typically performed at 1:1000 dilution overnight at 4°C, though optimization may be required for specific antibodies. For detection of both RRAS2 and downstream targets like phospho-ERK1/2, stripping and reprobing protocols should be carefully optimized to prevent signal loss. Loading controls such as GAPDH (14C10 Rabbit mAb) have been successfully used in RRAS2 Western blotting protocols. These modifications help ensure specific detection of RRAS2 protein while minimizing background and optimizing signal-to-noise ratio .

What are the key considerations for selecting appropriate RRAS2 antibodies for different research applications?

Selecting the appropriate RRAS2 antibody requires careful consideration of several key factors based on the specific research application. For Western blotting, antibodies validated specifically for this application should be chosen, with consideration of the epitope location—antibodies targeting conserved regions may be preferred for cross-species detection. Flow cytometry applications require conjugated antibodies (such as PE-conjugated EM-50 clone) optimized for intracellular staining, with recommendations for using 1-4 μg/mL concentration. For immunohistochemistry, antibodies validated for formalin-fixed, paraffin-embedded tissues should be selected, with consideration of retrieval methods compatible with RRAS2 epitopes. When studying RRAS2 variants or mutations, epitope location is critical—antibodies targeting regions distant from the mutation site are preferred to ensure detection regardless of the variant's effect on protein structure. For co-localization studies using immunofluorescence, consider host species compatibility with other primary antibodies in the panel to avoid cross-reactivity during secondary antibody detection. When examining RRAS2-specific effects versus other Ras family members, highly specific antibodies like the EM-50 clone that do not cross-react with R-Ras1, H-Ras, K-Ras, and N-Ras are essential. For studies involving both human and animal models, antibodies with validated cross-reactivity to relevant species (human, mouse, rat) should be selected to ensure consistent detection across experimental systems .

How can researchers troubleshoot weak or non-specific signals when using RRAS2 antibodies?

When encountering weak or non-specific signals with RRAS2 antibodies, systematic troubleshooting approaches can identify and resolve technical issues. For weak signals, researchers should first verify sample preparation protocols, ensuring complete cell lysis and protein extraction, particularly for membrane-associated proteins like RRAS2. Increasing protein concentration loaded per well or extending primary antibody incubation time (overnight at 4°C) may enhance detection sensitivity. For non-specific bands, optimizing blocking conditions by testing alternative blocking agents (5% BSA vs. non-fat dry milk) or increasing blocking time can reduce background. Adjusting antibody concentration through careful titration experiments can identify the optimal dilution that maximizes specific signal while minimizing background. Fresh preparation of detection reagents and verification of secondary antibody compatibility with the primary antibody host species are essential troubleshooting steps. For inconsistent results across experiments, standardizing lysate preparation protocols and including positive control samples with known RRAS2 expression can improve reproducibility. When studying post-translational modifications or activation states, phosphatase and protease inhibitors should be included in lysis buffers to preserve protein modifications. For persistent issues with monoclonal antibodies, testing alternative clones that recognize different epitopes may overcome problems with epitope masking or conformation-dependent recognition. These systematic approaches can substantially improve RRAS2 detection quality across various experimental applications .

How should researchers quantify and normalize RRAS2 expression data from Western blots?

Accurate quantification and normalization of RRAS2 expression data from Western blots requires rigorous methodological approaches. Researchers should employ digital image capture systems with linear dynamic range capabilities rather than film-based detection to ensure quantitative accuracy. Densitometric analysis using software like ImageJ, Image Lab, or specialized analysis platforms should include background subtraction to account for membrane or lane-specific background signals. For normalization, housekeeping proteins such as GAPDH have been successfully used as loading controls in RRAS2 studies, though researchers should verify the stability of expression across experimental conditions. The normalization calculation should divide the RRAS2 band intensity by the corresponding loading control intensity for each sample. When comparing RRAS2 expression across multiple blots, inclusion of a common internal reference sample on each blot allows for inter-blot normalization. For studies examining both RRAS2 and its downstream effectors like phospho-ERK1/2, normalization of phosphorylated protein to total protein provides a more accurate assessment of activation status than comparison to housekeeping proteins alone. Statistical analysis should include multiple biological replicates (minimum n=3) and appropriate statistical tests based on data distribution. Transparent reporting of quantification methods, including software settings, normalization strategy, and statistical approaches, is essential for reproducibility and proper interpretation of RRAS2 expression data .

What experimental controls are essential when studying RRAS2 activation in signaling pathways?

When studying RRAS2 activation in signaling pathways, a comprehensive set of experimental controls is essential for accurate interpretation. Positive controls should include cells or tissues with known high RRAS2 expression and activation, such as specific cancer cell lines, while negative controls might include RRAS2 knockdown or knockout systems generated through siRNA or CRISPR technologies. Technical controls should include primary antibody omission controls to assess secondary antibody specificity and isotype controls (e.g., IgG1 kappa for the EM-50 monoclonal antibody) to evaluate non-specific binding. For pathway activation studies, controls with known MAPK pathway activators (e.g., EGF stimulation) or inhibitors (e.g., MEK inhibitors like U0126) can validate assay functionality. When studying specific RRAS2 mutations such as those found in Noonan syndrome (p.Gly23Val and p.Gly24Glu), wild-type RRAS2 expression constructs serve as essential reference controls. Time-course experiments should include appropriate baseline (time zero) samples to account for basal activation levels. For overexpression studies, empty vector transfection controls are necessary to distinguish specific RRAS2 effects from transfection-related artifacts. These diverse controls collectively ensure that observed effects are specifically attributable to RRAS2 activity rather than experimental variables or non-specific interactions .

What are the potential pitfalls in interpreting RRAS2 expression data across different experimental systems?

Interpretation of RRAS2 expression data across different experimental systems presents several potential pitfalls that researchers must carefully navigate. Cross-species variations in RRAS2 sequence and expression patterns can complicate comparisons between human samples and animal models, despite high conservation (human RRAS2 shares significant homology with mouse ortholog). Antibody cross-reactivity with other Ras family members (R-Ras1, H-Ras, K-Ras, N-Ras) can lead to misinterpretation of expression data, necessitating highly specific antibodies like clone EM-50 that has been validated not to cross-react with these related proteins. Cell type-specific expression patterns and subcellular localization differences of RRAS2 may result in inconsistent detection across tissue types or cell lines, requiring careful validation in each system. The dynamic nature of RRAS2 activation states (GTP-bound versus GDP-bound) can affect epitope accessibility and antibody binding efficiency, potentially leading to underestimation of total protein levels. In disease models, particularly cancer and Noonan syndrome studies, mutations in RRAS2 may alter antibody binding characteristics, especially if the epitope includes or is near the mutation site. Overexpression systems may not recapitulate physiological regulation and can lead to artificial pathway activation, necessitating careful comparison with endogenous expression systems. Awareness of these potential pitfalls allows researchers to design appropriate controls and validation experiments to ensure accurate interpretation of RRAS2 expression data across diverse experimental contexts .

How can RRAS2 antibodies be used to investigate potential therapeutic approaches targeting RRAS2?

RRAS2 antibodies enable multiple strategies for investigating therapeutic approaches targeting this oncogenic protein. Target validation studies can employ RRAS2 antibodies in Western blotting and immunohistochemistry to verify protein expression in patient-derived samples, establishing clinical relevance before therapeutic development. For drug screening applications, RRAS2 antibodies can be used in high-throughput assays to evaluate how candidate compounds affect RRAS2 protein stability, localization, or downstream signaling. In mechanism-of-action studies, RRAS2 antibodies combined with phospho-specific antibodies for downstream effectors (like phospho-ERK1/2) can reveal how potential therapeutics modulate RRAS2-dependent signaling cascades. The validated mouse model of B-cell chronic lymphocytic leukemia (Rosa26-RRAS2 fl/fl-mb1-Cre), which demonstrates 100% penetrance of disease, provides an excellent system for testing RRAS2-targeted therapies, with antibodies enabling assessment of target engagement and pathway modulation. For developing direct RRAS2 inhibitors, structural insights from antibody epitope mapping may inform rational drug design approaches. In clinical trials, RRAS2 antibodies could serve as companion diagnostics to identify patients most likely to benefit from RRAS2-targeted therapies. These diverse applications highlight how RRAS2 antibodies contribute to the translational pipeline from basic discovery to therapeutic development, particularly in diseases where RRAS2 dysregulation plays a causal role, such as certain leukemias and Noonan syndrome .

What techniques can be used to study RRAS2 interactions with B and T cell receptors?

Investigation of RRAS2 interactions with B and T cell receptors requires sophisticated methodological approaches where RRAS2 antibodies play central roles. Co-immunoprecipitation (Co-IP) using RRAS2 antibodies can isolate protein complexes containing RRAS2 and B cell receptor (BCR) or T cell receptor (TCR) components, with subsequent Western blotting to identify specific interaction partners. Proximity ligation assays (PLA) provide spatial resolution of protein interactions in situ, visualizing RRAS2-BCR/TCR complexes within intact cells with subcellular localization information. For dynamic interaction studies, live-cell imaging with fluorescently tagged RRAS2 antibody fragments or RRAS2-GFP fusion proteins allows tracking of receptor interactions following immune synapse formation. FRET/FLIM (Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging) techniques can detect direct molecular interactions between RRAS2 and receptor components at nanometer-scale distances. Mass spectrometry analysis of RRAS2 immunoprecipitates can identify novel interaction partners and post-translational modifications regulating these interactions. Structure-function studies can employ RRAS2 mutants (particularly those affecting the GDP/GTP binding pocket) to investigate how nucleotide binding state affects receptor interactions, as previous research has shown that RRAS2 interacts preferentially with immunoreceptor tyrosine-based activation motifs (ITAMs) in its GDP-bound form. These methodologies collectively enable detailed characterization of how RRAS2 provides tonic survival signals through BCR/TCR interactions and regulates receptor internalization following immune synapse formation .

What emerging applications exist for studying RRAS2 in single-cell analysis and spatial transcriptomics?

Emerging technologies in single-cell analysis and spatial transcriptomics offer revolutionary approaches for studying RRAS2 biology with unprecedented resolution. Single-cell proteomics using mass cytometry (CyTOF) with metal-conjugated RRAS2 antibodies enables quantification of RRAS2 protein levels alongside dozens of other markers in individual cells, revealing heterogeneity within seemingly homogeneous populations. Imaging mass cytometry combines this high-parameter protein detection with spatial information, mapping RRAS2 expression patterns within tissue architecture at subcellular resolution. RNA-protein co-detection methods like CITE-seq can simultaneously measure RRAS2 protein (using antibody-oligonucleotide conjugates) and transcriptome-wide gene expression in the same cells, revealing potential discordance between RRAS2 mRNA and protein levels. Spatial transcriptomics approaches can map RRAS2 mRNA expression within tissue contexts, complementing protein-level detection with RRAS2 antibodies in adjacent sections. For live-cell applications, nanobody-based RRAS2 detection could enable dynamic tracking of protein localization and interactions in living systems. In disease models like the Rosa26-RRAS2 fl/fl-mb1-Cre mouse model of CLL, these technologies could reveal how RRAS2 overexpression creates cellular heterogeneity within the leukemic population and identify potential therapeutic vulnerabilities. Similarly, in Noonan syndrome models with RRAS2 pathogenic variants, single-cell approaches could elucidate how mutation-induced signaling changes affect specific cell populations during development. These cutting-edge applications represent the frontier of RRAS2 research, offering unprecedented insights into its roles in normal physiology and disease pathogenesis .