SPC24 Antibody, HRP conjugated

What is SPC24 and what cellular functions does it perform?

SPC24 (also known as Kinetochore protein Spc24, hSpc24, or SPBC24) functions as a critical component of the essential kinetochore-associated NDC80 complex. This complex is required for chromosome segregation and spindle checkpoint activity during cell division. SPC24 specifically contributes to kinetochore integrity and the organization of stable microtubule binding sites in the outer plate of the kinetochore. The NDC80 complex, which includes SPC24, synergistically enhances the affinity of the SKA1 complex for microtubules and may enable the NDC80 complex to track depolymerizing microtubules during mitosis. These functions make SPC24 a significant target for research in cell cycle regulation and mitotic processes .

What are the key specifications of commercially available SPC24 Antibody, HRP conjugated?

The SPC24 Antibody, HRP conjugated is typically a polyclonal antibody raised in rabbits using recombinant human Kinetochore protein Spc24 protein (amino acids 21-97) as the immunogen. The antibody demonstrates reactivity with human samples and is primarily validated for ELISA applications. It is supplied in liquid form with >95% purity, purified using Protein G affinity chromatography. The antibody is generally provided in a buffer containing preservatives such as 0.03% Proclin 300, with 50% glycerol and 0.01M PBS at pH 7.4. For optimal preservation of activity, the antibody should be stored at -20°C or -80°C, with researchers advised to avoid repeated freeze-thaw cycles .

What experimental applications is SPC24 Antibody, HRP conjugated suitable for?

SPC24 Antibody, HRP conjugated is primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications. The HRP (horseradish peroxidase) conjugation enables direct detection without requiring secondary antibodies, which simplifies experimental workflows and potentially reduces background signal in ELISA-based detection systems. While ELISA is the confirmed application, researchers should determine optimal dilutions through experimental titration, as sensitivity may vary based on specific experimental conditions, detection systems, and sample types. The HRP conjugation makes this antibody particularly suitable for colorimetric detection systems using appropriate substrates such as TMB (3,3',5,5'-tetramethylbenzidine) .

How should researchers properly store and handle SPC24 Antibody, HRP conjugated to maintain its activity?

For optimal preservation of SPC24 Antibody, HRP conjugated activity, researchers should aliquot the antibody upon receipt to minimize freeze-thaw cycles and store it at -20°C or -80°C. The HRP conjugate is sensitive to light exposure, so protection from light during storage and handling is essential. When working with the antibody, it should be thawed gently on ice and returned to storage promptly after use. Repeated freeze-thaw cycles can lead to denaturation and loss of activity of both the antibody and the HRP enzyme. Additionally, contamination should be prevented by using sterile technique when handling the antibody. For long-term storage considerations, researchers should verify the stability period provided by the manufacturer and avoid using the antibody beyond this timeframe .

How does the selection of recombinant human SPC24 protein (21-97AA) as immunogen affect epitope recognition and experimental outcomes?

The selection of amino acids 21-97 of human SPC24 as the immunogen has significant implications for epitope recognition and experimental applications. This region represents a substantial portion of the 197-amino acid full-length protein and likely contains immunodominant epitopes within the functional domains of SPC24. The targeted region may influence the antibody's ability to recognize SPC24 in different experimental contexts, particularly when the protein undergoes conformational changes during the cell cycle or interactions with other NDC80 complex components. Researchers should consider that this antibody may not recognize epitopes outside the 21-97AA region, which could affect detection in contexts where these regions are important. Additionally, post-translational modifications within this region might alter antibody recognition, potentially affecting experimental outcomes when studying dynamically regulated SPC24 .

What considerations should be made when using SPC24 Antibody, HRP conjugated for analyzing SPC24 expression in different phases of the cell cycle?

When analyzing SPC24 expression across different cell cycle phases using HRP-conjugated antibodies, researchers should consider several critical factors. First, SPC24 localization changes dramatically during mitosis as kinetochores assemble and disassemble, necessitating appropriate fixation methods that preserve kinetochore structures. Second, expression levels and phosphorylation states of SPC24 fluctuate throughout the cell cycle, potentially affecting epitope accessibility and antibody recognition. Researchers should employ cell synchronization techniques (e.g., double thymidine block, nocodazole treatment) to enrich populations at specific cell cycle stages for more accurate comparative analyses. Additionally, co-staining with cell cycle markers (e.g., pH3 for mitosis) would provide internal controls to correlate SPC24 detection with specific cell cycle phases. Finally, the HRP conjugation may cause steric hindrance at the kinetochore, potentially affecting binding efficiency in densely packed structures, which should be considered when interpreting negative results .

How can researchers validate the specificity of SPC24 Antibody, HRP conjugated in experimental systems?

To rigorously validate the specificity of SPC24 Antibody, HRP conjugated, researchers should implement multiple complementary approaches. First, perform side-by-side comparison with alternative SPC24 antibodies from different clones or sources in parallel experiments. Second, include appropriate negative controls such as IgG isotype controls matched to the host species (rabbit) and concentration. Third, conduct knockdown validation through siRNA or CRISPR-Cas9 targeting of SPC24, which should result in reduced or absent signal if the antibody is specific. Fourth, perform peptide competition assays using the immunizing peptide (amino acids 21-97 of human SPC24) to confirm binding specificity. For cell-based assays, include cell lines known to express different levels of SPC24 to demonstrate proportional signal intensity. Finally, researchers can perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down SPC24 and its known interacting partners in the NDC80 complex .

What methodological adaptations are necessary when transitioning from ELISA to potential immunohistochemistry applications with SPC24 Antibody, HRP conjugated?

Transitioning from ELISA to immunohistochemistry (IHC) applications with SPC24 Antibody, HRP conjugated requires several methodological adaptations. First, antigen retrieval optimization is critical, as formalin fixation can mask epitopes; researchers should test multiple retrieval methods (heat-induced with citrate buffer, Tris-EDTA, or enzymatic methods). Second, blocking procedures must be optimized to prevent non-specific binding, particularly important with polyclonal antibodies. Third, dilution optimization is essential, typically starting with higher concentrations (1:50-1:200) for IHC compared to ELISA (1:500-1:2000). Fourth, incubation conditions should be adjusted, often requiring longer incubation times (overnight at 4°C) for tissue penetration. Fifth, endogenous peroxidase activity must be quenched (using H₂O₂ treatment) to prevent false-positive signals from endogenous HRP-like activity in tissues. Finally, the detection system may need adjustment, as the direct HRP conjugation might provide insufficient signal amplification for low-abundance proteins in tissues, potentially requiring additional signal enhancement methods .

What are the optimal experimental conditions for using SPC24 Antibody, HRP conjugated in ELISA assays?

For optimal ELISA performance with SPC24 Antibody, HRP conjugated, researchers should consider the following methodological parameters. Begin with a coating concentration titration (typically 1-5 μg/ml) of recombinant SPC24 protein or cell lysates containing SPC24. Blocking should be performed with 3-5% BSA or non-fat milk in PBS/TBS with 0.05% Tween-20 for 1-2 hours at room temperature. For the primary antibody (SPC24 Antibody, HRP conjugated) incubation, start with a dilution range of 1:500-1:2000 as recommended, with overnight incubation at 4°C providing optimal sensitivity. After washing (4-6 times with PBS/TBS containing 0.05% Tween-20), proceed directly to substrate addition (TMB for HRP) without a secondary antibody step, as the antibody is already HRP-conjugated. Develop the reaction for 5-30 minutes (optimized empirically) before stopping with 2N H₂SO₄ or equivalent. Include appropriate negative controls (non-specific rabbit IgG-HRP at matching concentrations) and positive controls (validated SPC24-expressing samples). This methodology should be optimized for each specific experimental system to achieve the best signal-to-noise ratio .

What strategies can researchers employ to optimize the detection sensitivity when working with low-abundance SPC24 protein?

When dealing with low-abundance SPC24 protein, researchers can implement several strategies to enhance detection sensitivity. First, consider sample enrichment through immunoprecipitation or subcellular fractionation to concentrate SPC24 from the nuclear/kinetochore fraction prior to analysis. Second, optimize incubation conditions by extending primary antibody incubation time (overnight at 4°C) and using signal enhancement systems compatible with HRP, such as tyramide signal amplification (TSA). Third, reduce background interference by implementing more stringent blocking (5% BSA with 0.1-0.3% Triton X-100) and additional washing steps. Fourth, consider cell synchronization to enrich for mitotic cells where SPC24 is more abundantly expressed at kinetochores. Fifth, use enhanced chemiluminescent substrates with higher sensitivity for HRP detection. Finally, consider technological alternatives such as digital ELISA platforms (e.g., Simoa) which can provide much higher sensitivity than conventional ELISA. These combined approaches can significantly improve the detection of low-abundance SPC24 while maintaining specificity .

How should researchers approach experimental design when studying SPC24 interactions with other NDC80 complex components using the HRP-conjugated antibody?

When studying SPC24 interactions with other NDC80 complex components (NUF2, NDC80/HEC1, SPC25) using HRP-conjugated SPC24 antibody, researchers should implement a multi-faceted experimental design. Begin with co-immunoprecipitation studies, carefully considering that the HRP conjugation might sterically hinder protein-protein interactions; a parallel approach with unconjugated SPC24 antibody might be necessary for comparison. For proximity-based interaction studies, consider proximity ligation assays (PLA) using the HRP-conjugated SPC24 antibody alongside unconjugated antibodies against other NDC80 components. When analyzing complex assembly dynamics, synchronize cells at different mitotic stages to capture temporal interaction patterns. For quantitative analysis of complex stoichiometry, combine immunoprecipitation with quantitative mass spectrometry using SILAC or TMT labeling. Additionally, implement crosslinking strategies before immunoprecipitation to stabilize transient interactions. Finally, validate interactions through reciprocal pulldowns with antibodies against other NDC80 components. This comprehensive approach accounts for the unique characteristics of the HRP-conjugated antibody while providing robust evidence of SPC24 interactions within the NDC80 complex .

What are common sources of false positive and false negative results when using SPC24 Antibody, HRP conjugated, and how can researchers address them?

When using SPC24 Antibody, HRP conjugated, several factors can lead to false results. For false positives, common sources include: (1) cross-reactivity with structurally similar proteins, which can be addressed by validating with knockout/knockdown controls; (2) endogenous peroxidase activity in samples, mitigated by incorporating a peroxidase quenching step (3% H₂O₂ treatment); (3) inadequate blocking, resolved by optimizing blocking conditions (5% BSA or milk, longer incubation); and (4) non-specific binding of the polyclonal antibody, reduced by pre-adsorption against irrelevant proteins. For false negatives, key issues include: (1) epitope masking due to protein-protein interactions at the kinetochore, improved by testing various fixation and extraction methods; (2) degradation of HRP activity, prevented by proper storage and avoiding repeated freeze-thaw cycles; (3) inadequate antigen retrieval in fixed samples, addressed by optimizing retrieval methods; and (4) expression levels below detection threshold, enhanced using signal amplification techniques compatible with HRP detection systems .

How can researchers differentiate between specific SPC24 detection and background signals in complex experimental systems?

To differentiate specific SPC24 detection from background signals, researchers should implement a comprehensive validation strategy. First, include critical experimental controls: positive controls (cells overexpressing SPC24), negative controls (SPC24 knockdown/knockout cells), and technical controls (isotype-matched rabbit IgG-HRP at equivalent concentration). Second, perform titration experiments to identify the optimal antibody concentration where specific signal is maximized while background is minimized. Third, implement competitive inhibition assays using the immunizing peptide (21-97AA of SPC24) to confirm signal specificity - true SPC24 signals should be significantly reduced. Fourth, validate the expected subcellular localization pattern (concentrated at kinetochores during mitosis) and molecular weight (approximately 22 kDa) to confirm target specificity. Fifth, employ dual-labeling with alternative SPC24 antibodies or known interacting partners (SPC25, NDC80) to confirm colocalization. Finally, analyze signal kinetics throughout the cell cycle - authentic SPC24 signals should show characteristic temporal patterns, with enhanced kinetochore localization during prometaphase and metaphase .

What analytical methods are most appropriate for quantifying SPC24 expression levels using HRP-conjugated antibodies?

For quantifying SPC24 expression levels using HRP-conjugated antibodies, researchers should select analytical methods based on experimental objectives and sample types. For absolute quantification, sandwich ELISA with standard curves using recombinant SPC24 protein (covering the 21-97AA region) provides the most reliable results. This approach should include standard curve validation for linearity (R² > 0.98) across the physiological concentration range. For relative quantification across samples, colorimetric ELISA with normalization to total protein concentration or housekeeping proteins is appropriate. When analyzing complex samples, competitive ELISA may provide better specificity by measuring displacement of labeled SPC24. For single-cell analysis, researchers might adapt the HRP-conjugated antibody for flow cytometry using HRP-compatible fluorescent substrates, though this requires extensive validation. Data analysis should include background subtraction, normalization, and statistical evaluation of technical and biological replicates. When comparing SPC24 levels across experimental conditions, matched loading controls and consistent methodology are essential for meaningful quantitative comparisons .

How should researchers interpret discrepancies between results obtained with SPC24 Antibody, HRP conjugated and other detection methods for SPC24?

When facing discrepancies between results obtained with SPC24 Antibody, HRP conjugated and alternative detection methods, researchers should systematically evaluate several potential explanations. First, epitope availability may differ between methods - the HRP-conjugated antibody targets amino acids 21-97, while other antibodies might recognize different regions that may be differentially accessible in certain experimental conditions. Second, the HRP conjugation might sterically hinder antibody binding in certain contexts, particularly in densely packed structures like kinetochores. Third, different detection sensitivities between methods could explain quantitative discrepancies - HRP-based detection typically offers high sensitivity but potentially different dynamic range compared to fluorescence-based methods. Fourth, post-translational modifications of SPC24 might differentially affect epitope recognition between antibodies. To resolve these discrepancies, researchers should perform parallel validation experiments with multiple antibodies, consider orthogonal detection methods (mass spectrometry, RNA expression), and integrate findings from complementary approaches to develop a comprehensive understanding of true SPC24 biology rather than relying on a single antibody or detection method .

What methodological approach would be optimal for studying SPC24 phosphorylation states using SPC24 Antibody, HRP conjugated?

Studying SPC24 phosphorylation states using SPC24 Antibody, HRP conjugated requires a carefully designed methodological approach. First, researchers should determine if the 21-97AA immunogen region contains known or predicted phosphorylation sites, as this affects whether the antibody can recognize phosphorylated forms. For sample preparation, implement phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) during cell lysis to preserve phosphorylation states. Consider phospho-enrichment techniques such as phosphoprotein purification columns or titanium dioxide chromatography before antibody application. To differentiate phosphorylated from non-phosphorylated SPC24, perform parallel analyses with and without lambda phosphatase treatment. For detection, consider two-dimensional electrophoresis followed by immunoblotting with the HRP-conjugated antibody to separate phospho-isoforms by isoelectric point. Alternatively, implement Phos-tag™ SDS-PAGE to create mobility shifts for phosphorylated proteins. For validation, compare results with phospho-specific antibodies targeting known SPC24 phosphorylation sites, and consider mass spectrometry to identify specific phosphorylation sites and their stoichiometry in your experimental system .

How can researchers effectively employ SPC24 Antibody, HRP conjugated in studying chromosome segregation defects in cancer cell models?

For studying chromosome segregation defects in cancer cell models using SPC24 Antibody, HRP conjugated, researchers should implement a multi-dimensional approach. Begin with baseline characterization of SPC24 expression levels across a panel of cancer cell lines with known chromosome instability phenotypes using quantitative ELISA. Correlate expression with the frequency of mitotic errors observed through live-cell imaging. For mechanistic studies, combine RNAi-mediated SPC24 depletion with rescue experiments using wild-type or mutant SPC24 constructs, followed by antibody detection of expression levels. To connect SPC24 dysregulation with specific segregation defects, perform time-course experiments during mitotic progression, synchronizing cells and collecting samples at precise timepoints for ELISA analysis of SPC24 levels. For spatial analysis, complement ELISA data with immunofluorescence studies using unconjugated SPC24 antibodies to visualize kinetochore localization patterns in cells exhibiting segregation defects. Additionally, investigate correlations between SPC24 levels and responses to anti-mitotic drugs like taxanes or aurora kinase inhibitors in cancer models. This comprehensive strategy links quantitative SPC24 expression data from HRP-conjugated antibody assays with functional outcomes in chromosome segregation .

What experimental design is most appropriate for studying the interaction between SPC24 and microtubule dynamics using the HRP-conjugated antibody?

For studying SPC24's role in microtubule dynamics, a multi-tiered experimental design is optimal. Begin with in vitro reconstitution assays combining purified tubulin, microtubule-associated proteins, and immunoprecipitated NDC80 complex (using SPC24 Antibody without HRP conjugation, as the conjugate might interfere with complex activity). For quantitative analysis of SPC24 levels in different microtubule stability conditions, treat cells with microtubule-stabilizing (taxol) or -destabilizing (nocodazole) agents before lysis and ELISA using the HRP-conjugated antibody. To directly visualize SPC24-microtubule interactions, implement proximity ligation assays between SPC24 and tubulin in fixed cells, using the HRP-conjugated antibody with a compatible detection system. For functional studies, combine SPC24 knockdown with rescue experiments using wild-type or mutant SPC24 constructs, and quantify expression using the HRP-conjugated antibody in ELISA format. Additionally, employ cold-stable microtubule assays to assess kinetochore-microtubule attachment stability in cells with modified SPC24 levels. This comprehensive approach connects quantitative SPC24 data from ELISA with functional outcomes in microtubule dynamics, though limitations of using HRP-conjugated antibodies in certain applications should be considered .

What is the optimal approach for integrating data from SPC24 Antibody, HRP conjugated experiments with genomic and proteomic datasets?

Integrating data from SPC24 Antibody, HRP conjugated experiments with broader genomic and proteomic datasets requires a multifaceted analytical strategy. Begin by establishing quantitative standards for SPC24 protein levels using ELISA with recombinant protein standards, allowing absolute quantification that can be correlated with transcript levels from RNA-seq data. For integration with proteomic data, normalize SPC24 ELISA results to total protein concentration measured by BCA or Bradford assays to enable direct comparison with mass spectrometry-based proteomics. When examining post-translational modifications, correlate antibody-based detection with phospho-proteomic datasets, considering potential epitope masking effects. For functional genomics integration, combine SPC24 protein levels with CRISPR screen data targeting mitotic regulators to identify synthetic interactions. Additionally, implement computational approaches such as gene set enrichment analysis (GSEA) to contextualize SPC24 expression patterns within broader cellular pathways. Visualize integrated data using dimensionality reduction techniques like principal component analysis or t-SNE to identify patterns across multiple datasets. This integrative approach provides a systems-level understanding of SPC24 biology beyond what can be achieved with antibody-based detection alone .

What technical considerations should researchers address when adapting SPC24 Antibody, HRP conjugated protocols for high-throughput screening applications?

Adapting SPC24 Antibody, HRP conjugated protocols for high-throughput screening requires addressing several technical considerations to ensure data quality and reproducibility. First, optimize antibody concentration through careful titration experiments in the high-throughput format to determine the minimum concentration providing reliable signal-to-noise ratio, which conserves reagent and minimizes cost. Second, implement robust positive and negative controls on each plate (recombinant SPC24 protein and SPC24-knockout samples) to enable plate-to-plate normalization and quality control metrics. Third, assess edge effects and positional biases in microplate formats, potentially implementing randomized sample positioning or edge-well exclusion strategies. Fourth, optimize liquid handling parameters for consistent antibody dispensing, with calibration validation before each screening campaign. Fifth, standardize incubation conditions using temperature-controlled incubators rather than room temperature to minimize environmental variability. Sixth, establish clear statistical criteria for hit identification, incorporating Z-factor analysis to assess assay quality and replicate correlation coefficients to ensure reproducibility. Finally, implement an automation-compatible data management system to track sample metadata, experimental conditions, and results across large datasets. These considerations enable reliable adaptation of SPC24 antibody assays to high-throughput formats while maintaining data integrity .

How might emerging super-resolution microscopy techniques be optimized for use with SPC24 Antibody, HRP conjugated?

While HRP-conjugated antibodies are not traditionally used for fluorescence microscopy, emerging technologies offer potential adaptations for super-resolution applications with SPC24 Antibody, HRP conjugated. Researchers could employ tyramide signal amplification (TSA) protocols, where the HRP activity catalyzes deposition of fluorophore-conjugated tyramide in close proximity to the antibody binding site. This approach is compatible with super-resolution techniques like Structured Illumination Microscopy (SIM) and Stimulated Emission Depletion (STED) microscopy. For optimal resolution, researchers should implement a two-step protocol: first, detection with SPC24 Antibody, HRP conjugated at high dilution (1:2000-1:5000) to minimize background; second, brief tyramide reaction (2-5 minutes) to prevent over-amplification that could compromise resolution. Sample preparation requires careful optimization, including gentle fixation (2% paraformaldehyde) and extraction methods that preserve kinetochore ultrastructure while improving antibody accessibility. For multi-color imaging, sequential TSA reactions with spectral unmixing algorithms can reduce channel bleed-through. This approach could enable visualization of SPC24 distribution within the NDC80 complex at the kinetochore with nanometer-scale resolution, particularly valuable for studying structural rearrangements during microtubule attachment and tension sensing .

What emerging analytical methods might enhance the quantitative analysis of SPC24 using HRP-conjugated antibodies?

Emerging analytical technologies could significantly enhance quantitative analysis of SPC24 using HRP-conjugated antibodies. Digital ELISA platforms (Simoa, Quanterix) offer single-molecule detection capabilities, potentially improving sensitivity by 100-1000 fold over conventional ELISA, enabling detection of SPC24 in extremely limited samples like circulating tumor cells or needle biopsies. Microfluidic immunoassay systems could provide rapid, automated analysis with minimal sample consumption while maintaining sensitivity. Mass cytometry (CyTOF) could be adapted using metal-labeled anti-HRP antibodies as secondary reagents, enabling simultaneous analysis of SPC24 alongside dozens of other proteins at single-cell resolution. For spatial analysis, digital spatial profiling platforms could be optimized using the HRP-conjugated antibody with compatible detection systems to quantify SPC24 expression with spatial context in tissue sections. Machine learning algorithms could be implemented for automated signal quantification and pattern recognition across complex datasets. Additionally, barcoded antibody technologies might allow multiplexed detection of SPC24 alongside other NDC80 complex components in single samples. These emerging technologies offer potential for more sensitive, specific, and comprehensive quantitative analysis of SPC24 in complex biological systems .