RUFY3 Antibody

Available RUFY3 Antibody Types and Their Characteristics

RUFY3 antibodies are primarily available as polyclonal preparations derived from rabbit hosts, with some mouse-derived options also documented. The antibodies target various regions including N-terminal domains (AA 1-65), central regions (AA 243-352), and C-terminal portions of the RUFY3 protein .

These antibodies exhibit reactivity across multiple species including human, mouse, rat, and in some cases, a broader range including dog, cow, xenopus, and other vertebrates. This cross-reactivity profile must be carefully considered when designing experiments involving multiple species or comparing results across different model organisms .

Optimal Applications for RUFY3 Antibody Detection

The most validated applications for RUFY3 antibodies include Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF), with some antibodies also suitable for immunohistochemistry on paraffin-embedded sections (IHC-P) . For Western blot applications, RUFY3 antibodies typically provide optimal results at concentrations around 1.0 μg/mL, with HRP-conjugated secondary antibodies recommended at dilutions between 1:50,000 and 1:100,000 .

Technical variations of RUFY3 antibodies include unconjugated formats and conjugated versions with FITC, HRP, and biotin, allowing researchers to select the appropriate format based on their specific detection system and experimental design requirements .

Lentiviral-Mediated Manipulation of RUFY3 Expression

Recent research has employed sophisticated lentiviral constructs to investigate RUFY3 function in neuronal models. Four key lentiviral vectors have been validated for RUFY3 studies:

• Lentivirus-negative control 1 (LV-NC1)

• Lentivirus-Rufy3 shRNA (LV-shRNA) for knockdown

• Lentivirus-negative control 2 (LV-NC2)

• Lentivirus-Rufy3 (LV-Rufy3) for overexpression

The sequence elements of these lentiviral vectors typically incorporate Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LV-Rufy3) and hU6-MCS-CBh-gcGFP-IRES-puromycin (LV-shRNA) components. These constructs have successfully demonstrated specificity and efficacy in both in vitro and in vivo models of RUFY3 manipulation .

Time-Course Analysis of RUFY3 Expression in Pathological Conditions

Temporal expression patterns of RUFY3 have been characterized in subarachnoid hemorrhage models, revealing significant downregulation after SAH induction. RUFY3 expression reaches its lowest levels approximately 24 hours post-SAH, followed by gradual recovery over a one-week period . This temporal profile provides critical guidance for researchers designing intervention studies, suggesting that the 24-hour timepoint represents an optimal window for therapeutic interventions targeting RUFY3-related pathways .

Multi-Parameter Assessment of Neuronal Integrity

Comprehensive assessment of neuronal damage in RUFY3 studies requires simultaneous monitoring of multiple markers. Research protocols have established that decreased myelin basic protein (MBP) immunopositivity combined with increased neurofilament heavy chain (N52) immunopositivity serves as reliable indicators of neuronal axon damage in experimental models .

The quantification of these markers through immunofluorescence staining enables correlation with RUFY3 expression levels, providing insights into the protective or pathological roles of RUFY3 in neuronal injury contexts. Statistical significance in these studies has been established at p-values ranging from p<0.05 to p<0.001 .

Parallel Analysis of RUFY3 at mRNA and Protein Levels

Comprehensive characterization of RUFY3 dynamics requires assessment at both transcriptional and translational levels. The integration of RT-PCR for mRNA quantification with Western blotting for protein detection has revealed that RUFY3 downregulation occurs at both levels after SAH, with significant decreases observed as early as 6 hours for mRNA and sustained through 24 hours for protein expression .

This multi-level analysis approach provides crucial insights into the regulatory mechanisms controlling RUFY3 expression and helps distinguish between transcriptional repression and post-translational modifications or protein degradation pathways that might be targeted therapeutically .

RUFY3 and Small GTPase Functional Relationships

RUFY3 functions as an adapter protein for small GTPases, particularly within the Ras family, contributing to neuronal polarity maintenance. The interaction between RUFY3 and small GTPases represents a critical regulatory mechanism in neuronal development and response to injury .

Experimental evidence indicates that RUFY3 interacts specifically with Rab5(Q79L) through its carboxyl terminus, suggesting a role in endosomal trafficking or signaling pathways mediated by this GTPase . This interaction may be particularly relevant for understanding RUFY3's function in neuronal polarity and axonal development.

Pharmacological Modulation of RUFY3-GTPase Pathways

Investigational approaches have employed GTPase modulators such as 8-pCPT-2'-O-Me-cAMP (8p-CPT), a Rap1 agonist, to probe the relationship between RUFY3 and specific GTPase pathways. Intriguingly, while 8p-CPT influences certain downstream effects, it does not significantly alter RUFY3 expression levels, suggesting parallel rather than directly sequential signaling pathways .

This finding highlights the complexity of RUFY3 regulation and indicates that therapeutic approaches targeting RUFY3 might need to focus on direct modulation rather than indirect approaches through GTPase activation pathways .

Therapeutic Potential of RUFY3 Modulation

These findings position RUFY3 as a potential therapeutic target in neurological conditions involving acute brain injury, with antibody-based detection methods serving as critical tools for validating and monitoring such interventions in both preclinical and translational research contexts .

Practical Considerations for RUFY3 Detection in Complex Tissue Samples

The detection of RUFY3 in heterogeneous brain tissue presents technical challenges that require optimization of antibody-based protocols. Immunohistochemistry on paraffin-embedded sections (IHC-P) represents a validated approach for RUFY3 detection in fixed brain tissue samples, with polyclonal antibodies showing reliable reactivity against both human and mouse targets .

For researchers working with tissue samples, antibodies purified by antigen-affinity chromatography have demonstrated superior performance, particularly when targeting recombinant protein sequences within the central region of human RUFY3 .