Snaclec rhodocytin subunit alpha Antibody, HRP conjugated

What is the molecular structure of rhodocytin and how does it relate to its function?

Rhodocytin is a C-type lectin-like protein from the venom of Calloselasma rhodostoma (Malayan pit viper) that forms a non-disulfide linked (αβ)₂ tetramer in its native state. The protein consists of α and β subunits that assemble into a heterooctamer with four α- and β-subunits . This quaternary structure is critical for its biological activity, as it creates a concave binding surface that is highly complementary to its target receptor, CLEC-2 (C-type lectin domain family 1 member B) . The higher-order multimericity of rhodocytin promotes clustering of CLEC-2 receptors on the platelet surface, which significantly enhances signal transduction activity compared to what an isolated αβ-heterodimer could achieve .

What are the validated applications for the HRP-conjugated Snaclec rhodocytin subunit alpha antibody?

The HRP-conjugated antibody has been validated primarily for ELISA applications . While the non-conjugated version has been validated for Western blotting applications as well , researchers should conduct preliminary validation tests when applying the HRP-conjugated version to other techniques. The antibody demonstrates high specificity for Calloselasma rhodostoma rhodocytin alpha subunit and can be used at the following dilutions:

What are the optimal conditions for using this antibody in Western blot applications?

While specific protocols for the HRP-conjugated version may require optimization, the non-conjugated version has been successfully used in Western blot at a concentration of 3µg/ml . For secondary detection, a goat polyclonal to rabbit IgG at 1/50000 dilution has been effective. The predicted band size for rhodocytin subunit alpha is 31 kDa, which matches the observed band size in validation studies .

When adapting these conditions for the HRP-conjugated version, researchers should:

• Eliminate the secondary antibody step

• Optimize blocking conditions to reduce background

• Adjust exposure time to accommodate the direct HRP signal

• Consider using enhanced chemiluminescence (ECL) substrates with appropriate sensitivity

How can the rhodocytin antibody be used to study the CLEC-2 signaling pathway in platelets?

The rhodocytin antibody can be instrumental in studying the CLEC-2 signaling pathway through several methodological approaches:

• Immunoprecipitation coupled with mass spectrometry: This technique allows researchers to pull down rhodocytin and its binding partners from platelet lysates, helping to identify components of the signaling complex . This approach has been successfully used to identify antigenic proteins in snake venoms.

• Inhibition studies: Pre-incubating platelets with the antibody can block rhodocytin-induced platelet aggregation, enabling researchers to study the specificity of CLEC-2-mediated activation .

• Fluorescence microscopy: Using the antibody in conjunction with fluorescently-labeled antibodies against downstream signaling molecules (Syk, PLC-gamma-2) can help visualize the spatiotemporal dynamics of signal propagation following CLEC-2 activation.

• Phosphoproteomic analysis: The antibody can be used to trigger CLEC-2 signaling, followed by analysis of tyrosine phosphorylation patterns to map the signaling cascade .

What are the critical residues in rhodocytin alpha subunit for CLEC-2 binding, and how can the antibody be used to study these interactions?

Research has identified Asp4 in the α-subunit of rhodocytin as critical for CLEC-2 binding . The HRP-conjugated antibody can be used to study these interactions through:

• Competitive binding assays: Researchers can use site-directed mutagenesis to create rhodocytin variants with alterations at Asp4 and other potentially important residues, then use the antibody in competitive binding assays to assess how these mutations affect CLEC-2 recognition.

• Epitope mapping: By determining whether the antibody's binding is affected by mutations at Asp4, researchers can assess whether this residue is part of the antibody's epitope, which could have implications for interpreting functional studies.

• Structure-function studies: The antibody can be used in conjunction with crystallography and other structural biology techniques to validate the importance of specific residues in the rhodocytin-CLEC-2 interaction.

What are common issues encountered when using the HRP-conjugated antibody in immunoassays?

Several methodological challenges may arise when using this antibody:

• Background signal: The HRP conjugation may lead to higher background in some applications. This can be addressed by:

  • Optimizing blocking solutions (try 3-5% BSA in PBS with 0.1% Tween-20)

  • Increasing wash steps (5-6 washes of 5 minutes each)

  • Diluting the antibody further (start with 1:5000 and adjust as needed)

• Signal intensity variation: HRP activity can be affected by storage conditions and buffer components. To ensure reproducible results:

  • Store the antibody at -20°C or -80°C and avoid repeated freeze-thaw cycles

  • Prepare fresh dilutions for each experiment

  • Include positive controls with known concentrations of target protein

• Cross-reactivity: While the antibody is specific for Calloselasma rhodostoma, researchers working with venoms from multiple snake species should validate specificity:

  • Include appropriate negative controls

  • Consider pre-absorption with related proteins if cross-reactivity is observed

How can the rhodocytin antibody be used in combination with other antibodies to study complex signaling pathways?

For multiplexed detection strategies:

• Sequential immunoprecipitation: When studying protein complexes, first immunoprecipitate with the rhodocytin antibody, then probe the precipitate with antibodies against suspected binding partners like CLEC-2, Syk, or PLC-gamma-2.

• Dual labeling immunofluorescence: If using the antibody for microscopy:

  • Use appropriate spectral separation between fluorophores

  • Control for potential cross-reactivity between antibodies

  • Consider spectral unmixing for closely overlapping signals

• Phospho-specific detection: When analyzing signaling cascades:

  • Use phospho-specific antibodies against Syk, PLC-gamma-2, and other downstream effectors

  • Employ a time course to capture the kinetics of signal propagation

  • Consider phosphatase inhibitors in lysis buffers to preserve phosphorylation states

How can the rhodocytin antibody be used to investigate thrombotic disorders?

The antibody can be applied in several sophisticated experimental approaches:

• Ex vivo platelet function assays: Using the antibody to detect rhodocytin-induced platelet activation in patient samples can help assess CLEC-2 pathway integrity in thrombotic disorders.

• Thrombosis models: In experimental thrombosis models, the antibody can be used to:

  • Detect rhodocytin localization within thrombi

  • Block rhodocytin-CLEC-2 interactions to assess contribution to thrombus formation

  • Evaluate rhodocytin as a potential biomarker for thrombotic risk

• Drug development screening: The antibody can be utilized in high-throughput screens to identify compounds that disrupt rhodocytin-CLEC-2 interaction as potential antithrombotic agents.

What is the role of rhodocytin in cancer metastasis research, and how can the antibody contribute to this field?

Rhodocytin has significant implications for cancer research due to its interaction with CLEC-2, which is a physiological binding partner of podoplanin (PDPN) . PDPN is expressed on certain tumor cells and plays a role in tumor cell-induced platelet aggregation and metastasis.

Research methodologies using the antibody include:

• Metastasis inhibition studies: Engineered mutant forms of rhodocytin have been shown to block CLEC-2/podoplanin-dependent platelet aggregation and experimental lung metastasis . The antibody can be used to detect and quantify these interactions.

• Tissue microarray analysis: The antibody can help assess CLEC-2 expression patterns in tumor samples and correlate with metastatic potential.

• In vivo metastasis models: The antibody can be used to track rhodocytin-mediated interactions in experimental metastasis models, potentially revealing new therapeutic targets.

How might rhodocytin research contribute to the development of novel antiplatelet or antimetastatic therapeutics?

Current research suggests several promising therapeutic applications:

• Inhibitory rhodocytin mutants: Studies have shown that certain rhodocytin mutants, such as αWTβK53A/R56A, can form heterotetramers that bind to CLEC-2 without inducing platelet aggregation . These mutants can block CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis .

• Structure-guided drug design: The crystal structure of rhodocytin at 2.4 Å resolution provides valuable structural information for designing small molecule inhibitors targeting the rhodocytin-CLEC-2 interaction .

• Therapeutic antibody development: The epitope information and binding characteristics of research antibodies against rhodocytin can inform the development of therapeutic antibodies targeting this pathway.

What are the comparative advantages of using rhodocytin versus other CLEC-2 activators in experimental systems?

Rhodocytin offers several methodological advantages for researchers:

• Receptor specificity: Unlike some platelet agonists that activate multiple pathways, rhodocytin specifically targets CLEC-2, allowing for cleaner experimental systems to study this pathway in isolation .

• Dose-dependent effects: Rhodocytin exhibits dose-dependent platelet activation, making it suitable for quantitative studies of CLEC-2 signaling thresholds .

• Structural insights: The well-characterized structure of rhodocytin provides context for interpreting experimental results in terms of specific molecular interactions .

• Cross-species reactivity: Rhodocytin activates CLEC-2 in both human and mouse platelets, facilitating translational research between animal models and human systems .