Snaclec rhodocytin subunit alpha Antibody, Biotin conjugated

What is Snaclec rhodocytin subunit alpha and what is its biological significance?

Snaclec rhodocytin subunit alpha (also known as Aggretin alpha chain or Rhodoaggretin subunit alpha) is a protein derived from the venom of Calloselasma rhodostoma (Malayan pit viper). Its primary biological function involves eliciting platelet aggregation through binding to the C-type lectin domain family 1 member B (CLEC1B/CLEC2). This binding interaction initiates a signaling cascade that leads to tyrosine phosphorylation in the cytoplasmic tail of CLEC1B, which subsequently promotes the binding of spleen tyrosine kinase (Syk). This process continues with the activation of PLC-gamma-2, ultimately resulting in platelet activation and aggregation . The protein belongs to the snaclec (snake C-type lectin) family and represents an important model for studying platelet activation mechanisms .

What are the key specifications of the biotin-conjugated Snaclec rhodocytin subunit alpha antibody?

The biotin-conjugated Snaclec rhodocytin subunit alpha antibody (product code CSB-PA888242LD01CBG) is a polyclonal antibody raised in rabbits against a recombinant immunogen comprising amino acids 1-136 of the Calloselasma rhodostoma Snaclec rhodocytin subunit alpha protein . The antibody has an IgG isotype and is supplied in liquid form with >95% purity following Protein G purification . Its storage buffer contains 0.03% Proclin 300 as a preservative, along with 50% glycerol and 0.01M PBS at pH 7.4 . The antibody has been validated for ELISA applications, with some variants also tested for Western blotting with recommended dilutions of 1/500-1/5000 .

What is the amino acid sequence of the recombinant Snaclec rhodocytin subunit alpha protein?

The full amino acid sequence of the recombinant Snaclec rhodocytin subunit alpha protein (residues 1-136) is:

GLEDCDFGWSPYDQHCYQAFNEQKTWDEAEKFCRAQENGAHLASIESNGEADFVSWLISQKDELADEDYWIGLRAQNKEQQCSSEWSDGSSVSYENLIDLHTKKC GALEKLTGFRKWVNYYCE QMHAFVCKLLPY

This sequence corresponds to the immunogen used to generate the antibody and represents the full-length protein expressed in Escherichia coli with >90% purity, suitable for SDS-PAGE analysis .

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal antibody activity, the biotin-conjugated Snaclec rhodocytin subunit alpha antibody should be stored at -20°C or -80°C immediately upon receipt . For long-term storage, researchers should aliquot the antibody to minimize the number of freeze-thaw cycles, as repeated freezing and thawing can significantly compromise antibody stability and performance . The antibody is supplied in a buffer containing 50% glycerol, which helps maintain protein stability during freeze-thaw transitions. When handling the antibody, it should be kept on ice and returned to -20°C promptly after use to preserve its activity and binding specificity .

How can researchers optimize ELISA protocols using this biotin-conjugated antibody?

When optimizing ELISA protocols with the biotin-conjugated Snaclec rhodocytin subunit alpha antibody, researchers should consider several methodological approaches. First, establish an appropriate coating concentration of the target antigen (typically 1-10 μg/mL) in carbonate/bicarbonate buffer (pH 9.6). After overnight incubation at 4°C, block non-specific binding sites with a protein-containing buffer such as 1-5% BSA or non-fat milk in PBS-T. For the antibody incubation step, prepare a titration series (typically starting at 1:500 and performing 2-fold serial dilutions) to determine optimal working concentration .

The biotin conjugation provides enhanced signal amplification when used with streptavidin-HRP detection systems. For optimal results, incorporate a streptavidin-HRP incubation step (typically at 1:5000-1:20000 dilution) followed by TMB substrate addition. Include appropriate positive and negative controls to validate specificity, and consider performing replicate measurements to ensure reproducibility. The assay sensitivity can be further enhanced by optimizing incubation times and temperatures, with typical parameters being 1-2 hours at room temperature or overnight at 4°C for the primary antibody incubation step .

What experimental considerations are important when studying the interaction between rhodocytin and CLEC1B/CLEC2?

When investigating the interaction between rhodocytin and CLEC1B/CLEC2, researchers should consider several experimental approaches. Co-immunoprecipitation assays using the biotin-conjugated antibody can help verify direct binding interactions. For functional studies examining the downstream signaling events, phosphorylation-specific antibodies targeting the cytoplasmic tail of CLEC1B and Syk kinase should be incorporated to monitor activation status .

Flow cytometry can be used to assess binding of rhodocytin to platelets expressing CLEC1B/CLEC2, while platelet aggregation assays provide functional readouts of pathway activation. When designing these experiments, it is critical to include appropriate controls such as CLEC1B/CLEC2 knockout or knockdown samples, blocking antibodies against CLEC1B/CLEC2, and inhibitors of downstream signaling components like Syk kinase inhibitors .

The controversial binding of rhodocytin to GPIbalpha (GP1BA) and alpha-2/beta-1 (ITGA2/ITGB1) integrins should be addressed using competitive binding assays, blocking antibodies specific for these receptors, and receptor-specific knockout models. These approaches will help distinguish the primary CLEC1B/CLEC2-mediated effects from potential secondary binding interactions .

How can researchers use this antibody to investigate the Syk-dependent signaling pathway in platelet activation?

To investigate the Syk-dependent signaling pathway in platelet activation using this antibody, researchers should design experiments that capture the sequential events following rhodocytin binding to CLEC1B/CLEC2. Begin by using the biotin-conjugated antibody in immunofluorescence or flow cytometry studies to confirm the presence and distribution of rhodocytin on the platelet surface. Then, employ phospho-specific antibodies to monitor the tyrosine phosphorylation events in the cytoplasmic tail of CLEC1B following rhodocytin binding .

For temporal analysis of pathway activation, conduct time-course experiments measuring Syk recruitment and phosphorylation status at multiple time points after rhodocytin exposure. Use pull-down assays with the biotin-conjugated antibody to isolate rhodocytin-receptor complexes, followed by Western blotting to identify associated signaling proteins. Incorporate specific inhibitors of Syk (e.g., R406, PRT318) to confirm pathway dependence, and use phospho-specific antibodies against PLC-gamma-2 to monitor downstream events .

For functional readouts, measure calcium mobilization, platelet aggregation, and granule secretion following rhodocytin exposure in the presence or absence of Syk inhibitors. Complement these approaches with genetic models (Syk-deficient platelets or CLEC1B/CLEC2 knockouts) to definitively establish the role of this signaling axis in rhodocytin-induced platelet activation .

What are common sources of background signal when using biotin-conjugated antibodies and how can they be minimized?

When working with biotin-conjugated antibodies like the Snaclec rhodocytin subunit alpha antibody, several common sources of background signal can compromise experimental results. Endogenous biotin in biological samples can compete with the biotinylated antibody for streptavidin binding, particularly in tissues with high biotin content (e.g., liver, kidney). To minimize this, researchers should pre-block samples with avidin/streptavidin followed by free biotin before adding the biotinylated antibody .

Non-specific binding to Fc receptors can be reduced by including an Fc receptor blocking reagent in the antibody diluent. Additionally, insufficient blocking or inappropriate blocking agents can lead to high background; optimize blocking conditions using different agents (BSA, normal serum, commercial blocking buffers) and concentrations (1-5%) .

Over-development of signal in chromogenic detection systems will increase background; carefully monitor reaction development and stop the reaction at the appropriate time. Cross-reactivity with other proteins can be assessed using pre-adsorption controls with the immunizing peptide. Finally, excessive antibody concentration will increase non-specific binding; perform titration experiments to determine the optimal antibody concentration that provides specific signal with minimal background .

How can researchers validate antibody specificity in snake venom protein research?

Validating antibody specificity is crucial in snake venom protein research due to the complex mixture of structurally similar proteins present in venoms. For the Snaclec rhodocytin subunit alpha antibody, researchers should employ a multi-faceted validation approach. Begin with Western blot analysis using crude venom and purified rhodocytin, expecting a band at the appropriate molecular weight (~18-20 kDa for the alpha subunit). Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide before use; specific signals should be significantly reduced or eliminated .

Cross-reactivity testing against venoms from closely related snake species can help establish specificity within the snaclec family. Immunoprecipitation followed by mass spectrometry analysis can confirm that the antibody is capturing the intended target. For functional validation, demonstrate that the antibody can block or detect the biological activity of rhodocytin in platelet aggregation assays .

Additionally, use knockout/knockdown controls where possible, or compare results with alternative antibodies targeting different epitopes of the same protein. Document all validation steps thoroughly, including positive and negative controls, to ensure reproducibility and reliability of subsequent experiments using this antibody .

What strategies can help overcome challenges in detecting low-abundance targets with this antibody?

Detecting low-abundance targets with the biotin-conjugated Snaclec rhodocytin subunit alpha antibody requires optimized protocols that maximize signal while maintaining specificity. The biotin-streptavidin detection system already provides signal amplification, but researchers can further enhance sensitivity through several approaches. Consider using tyramide signal amplification (TSA), which can increase sensitivity by 10-100 fold through catalytic deposition of multiple biotin or fluorophore molecules .

Sample enrichment techniques such as immunoprecipitation or affinity purification before analysis can concentrate the target protein. Extend primary antibody incubation times (overnight at 4°C) to allow maximal binding, and optimize detection reagent concentrations and incubation parameters. For Western blotting, use highly sensitive chemiluminescent substrates or near-infrared fluorescent detection systems .

In ELISA applications, consider sandwich ELISA formats where the target is captured by one antibody and detected by the biotin-conjugated antibody, potentially improving both specificity and sensitivity. Use larger sample volumes when possible, and reduce washing stringency slightly (without compromising specificity) to retain more bound antibody. Finally, integrate signal over longer exposure times in imaging applications, and utilize image analysis software for quantification of weak signals against background .

How might this antibody contribute to comparative studies of C-type lectins across different snake species?

The Snaclec rhodocytin subunit alpha antibody can serve as a valuable tool for comparative studies of C-type lectins across different snake species, particularly within the viperidae family. Researchers can use this antibody in cross-reactivity studies to assess conservation of epitopes among C-type lectins from different species. Begin by performing Western blot or ELISA analysis of venom samples from multiple snake species, quantifying relative signal intensities to determine epitope conservation and evolutionary relationships .

For more detailed analysis, coupled with proteomics approaches, the antibody can help identify and isolate novel C-type lectins for subsequent characterization. Immunohistochemistry of venom glands from different species using this antibody can reveal patterns of C-type lectin expression and storage. Cross-neutralization studies can assess whether the antibody inhibits the biological activities of C-type lectins from other species, providing insights into structural and functional conservation .

By combining these approaches with phylogenetic analysis, researchers can construct evolutionary relationships among snake venom C-type lectins and correlate structural conservation with functional properties. This comprehensive approach will advance our understanding of the evolution and diversification of this important protein family across snake species .

What are the potential applications of this antibody in studying novel platelet activation pathways?

The biotin-conjugated Snaclec rhodocytin subunit alpha antibody offers several applications for studying novel platelet activation pathways beyond the established CLEC1B/CLEC2 signaling axis. Researchers can use the antibody in affinity purification experiments to identify novel binding partners of rhodocytin on the platelet surface, followed by mass spectrometry to characterize these interactions .

Proximity ligation assays (PLA) using this antibody paired with antibodies against candidate receptors can reveal spatial relationships and potential interactions on the platelet surface. The antibody can also be employed in competitive binding studies to investigate the controversial interactions with GPIbalpha and alpha-2/beta-1 integrins, helping to resolve conflicting data in the literature .

For mechanistic studies, the antibody can be used to monitor the localization and clustering of rhodocytin during platelet activation using super-resolution microscopy. When combined with phosphoproteomic analysis, this approach can identify novel downstream signaling events triggered by rhodocytin binding. Additionally, the antibody can be utilized in screening assays to identify small molecule inhibitors of rhodocytin-receptor interactions, potentially leading to new therapeutic approaches for thrombotic disorders .

How should researchers interpret conflicting data about rhodocytin-induced platelet aggregation pathways?

When confronting conflicting data regarding rhodocytin-induced platelet aggregation pathways, researchers should apply a systematic analytical approach. The literature contains controversial findings about rhodocytin binding to GPIbalpha (GP1BA) and alpha-2/beta-1 (ITGA2/ITGB1) integrins in addition to the well-established CLEC1B/CLEC2 pathway . To reconcile these discrepancies, first consider methodological differences between studies, including protein preparation methods, concentration ranges, and assay conditions.

Perform concentration-dependent studies to determine if different receptors engage at different rhodocytin concentrations. Use receptor-specific knockout models or blocking antibodies to assess the contribution of each potential receptor to the aggregation response. Examine the temporal sequence of signaling events to determine if certain pathways are primary while others represent secondary effects .

Consider species differences in receptor expression and function, as studies using platelets from different species may yield varying results. Evaluate the oligomeric state of rhodocytin in different experimental conditions, as this may affect receptor binding preferences. Finally, use the biotin-conjugated antibody in competitive binding assays to directly assess the relative contribution of different receptors to rhodocytin binding under controlled conditions .

What quantitative approaches can be used to analyze binding kinetics of rhodocytin to CLEC1B/CLEC2?

To analyze the binding kinetics of rhodocytin to CLEC1B/CLEC2, researchers can employ several quantitative biophysical techniques. Surface Plasmon Resonance (SPR) represents a powerful approach, where purified CLEC1B/CLEC2 is immobilized on a sensor chip and varying concentrations of rhodocytin are flowed over the surface. The biotin-conjugated antibody can be used to detect bound rhodocytin, generating sensorgrams that yield association (kon) and dissociation (koff) rate constants, as well as the equilibrium dissociation constant (KD) .

Bio-Layer Interferometry (BLI) offers similar kinetic information and can utilize the biotin-conjugated antibody immobilized on streptavidin sensors to capture rhodocytin, followed by exposure to soluble CLEC1B/CLEC2. Isothermal Titration Calorimetry (ITC) provides complementary thermodynamic parameters (ΔH, ΔS, ΔG) in addition to binding constants, offering insights into the energetic basis of the interaction .

For cellular systems, fluorescence-based approaches such as Fluorescence Resonance Energy Transfer (FRET) between labeled rhodocytin and CLEC1B/CLEC2 can monitor binding in real-time. Microscale Thermophoresis (MST) offers an alternative method requiring small sample volumes. The resulting quantitative binding parameters can be integrated into computational models of platelet activation to understand how rhodocytin binding kinetics influence downstream signaling dynamics .