SPC24 Antibody, Biotin conjugated

What is SPC24 and what cellular functions does it perform?

SPC24 (also known as SPBC24 or hSpc24) functions as an essential component of the kinetochore-associated NDC80 complex, which plays a critical role in chromosome segregation and spindle checkpoint activity. Specifically, SPC24 is required for maintaining kinetochore integrity and organizing stable microtubule binding sites in the outer plate of the kinetochore . The NDC80 complex, containing SPC24, synergistically enhances the affinity of the SKA1 complex for microtubules and may enable the NDC80 complex to track depolymerizing microtubules during cell division . This function is fundamentally important for proper chromosomal segregation during mitosis, with dysregulation potentially contributing to genomic instability and cancer development.

How does biotin conjugation enhance SPC24 antibody utility in research applications?

Biotin conjugation of SPC24 antibodies leverages the exceptionally strong non-covalent interaction between biotin and avidin/streptavidin (Kd ≈ 10^-15 M), creating a versatile detection system for various assays. The avidin-biotin ELISA methodology provides significant advantages over traditional radioactive antigen-binding assays (RABA), offering reduced hazard potential while maintaining high sensitivity . In practical terms, biotin-conjugated SPC24 antibodies enable the detection of nanogram-per-milliliter quantities of target protein with reproducibility rates correlating with standard methods at approximately 76% . Additionally, biotin conjugation permits multiplexed detection strategies when combined with appropriately labeled avidin/streptavidin conjugates, enhancing experimental flexibility.

What experimental applications are most suitable for biotin-conjugated SPC24 antibodies?

Based on available research data, biotin-conjugated SPC24 antibodies excel in ELISA applications, particularly when employing avidin-coated plates for consistent capture . These antibodies can be utilized effectively for detecting SPC24 in human samples, with demonstrated applications in ELISA systems . For researchers requiring more specialized applications, the biotin conjugation confers compatibility with immunohistochemistry (IHC) techniques when paired with appropriate visualization systems (e.g., streptavidin-HRP). While Western blotting applications have been established for unconjugated SPC24 antibodies, the biotin-conjugated versions may require additional optimization for this application.

What sample preparation protocols maximize SPC24 detection using biotin-conjugated antibodies?

For optimal SPC24 detection using biotin-conjugated antibodies, researchers should implement a systematic sample preparation approach. Cell or tissue lysates should be prepared in buffers containing appropriate protease inhibitors to prevent degradation of the target protein. For ELISA applications, avidin-coated plates provide superior consistency compared to direct coating with capture antibodies . When working with capsular polysaccharides or similar antigens, derivatization using adipic acid dihydrazide (ADH) prior to biotinylation preserves antigenic epitopes while enhancing binding characteristics . This methodological approach ensures consistent antibody performance while maintaining native protein conformation for accurate detection.

How should researchers determine optimal concentration ranges for biotin-conjugated SPC24 antibodies?

Optimization of biotin-conjugated SPC24 antibody concentration requires systematic titration experiments. Based on available technical data, Western blotting applications typically employ dilution ranges of 1:500-1:2000 for optimal results . For ELISA applications, preliminary titration experiments should establish standard curves using known concentrations of purified SPC24 protein. Antibody concentrations should be adjusted to achieve a balance between sensitivity (signal amplitude) and specificity (signal-to-noise ratio). Optimization should account for the specific biotin conjugation ratio, as higher biotin incorporation can sometimes lead to reduced antigen recognition or increased non-specific binding.

What detection systems provide optimal sensitivity with biotin-conjugated SPC24 antibodies?

Avidin-biotin detection systems offer superior sensitivity for SPC24 quantification. Research demonstrates that avidin-coated plates provide consistent binding surfaces for biotinylated proteins, avoiding the inconsistent binding issues observed with direct plate coating . For enhanced sensitivity in chromogenic applications, streptavidin-HRP conjugates with amplification substrates (such as tyramide) can significantly lower detection thresholds. Fluorescence-based detection using streptavidin-fluorophore conjugates provides multiplexing capabilities and potentially higher sensitivity depending on instrumentation. Importantly, endogenous biotin blocking steps should be incorporated when working with biotin-rich tissues to minimize background interference.

How does SPC24 expression correlate with cancer progression and clinical parameters?

SPC24 expression demonstrates significant correlation with clinical parameters across multiple cancer types. In prostate cancer specifically, increased SPC24 expression is associated with patients older than 60 years compared to younger patients, elevated prostate-specific antigen (PSA) levels (P<0.05), and lymph node metastasis (P<0.05) . Higher SPC24 expression correlates with negative clinical outcomes in prostate cancer patients (P<0.05) . The table below summarizes key correlations between SPC24 expression and clinicopathological features in prostate cancer:

Notably, SPC24 expression is significantly elevated in prostatitis, benign prostatic hypertrophy (BPH), and prostate cancer compared to adjacent/normal tissues . High SPC24 expression particularly associates with advanced Gleason stages (IV and V; P<0.05) , suggesting utility as a prognostic biomarker.

How can SPC24 expression data be integrated with other molecular markers?

Integration of SPC24 expression data with other molecular markers enhances diagnostic and prognostic value. Research indicates that combining SPC24 with NDC80 (an SPC24 protein interaction partner) and BUB1 (a core subunit of the spindle assembly checkpoint) provides improved diagnostic capability for prostate cancer compared to individual markers . Binary logistic regression algorithms combining receiver operating characteristic data between SPC24 and BUB1 or NDC80 demonstrate superior diagnostic performance over traditional prostate cancer markers . Gene Ontology and pathway functional enrichment analysis further suggests that NDC80 and BUB1 associate with SPC24 in prostate cancer development . This integrated approach allows researchers to develop more comprehensive molecular signatures with enhanced clinical utility.

What techniques can effectively evaluate SPC24 interactions with other NDC80 complex components?

Advanced research into SPC24 interactions requires sophisticated methodological approaches. Co-immunoprecipitation (Co-IP) using biotin-conjugated SPC24 antibodies, followed by mass spectrometry analysis, provides comprehensive identification of protein interaction partners. Evidence demonstrates successful immunoprecipitation of SPC24 from cell lysates using specific antibodies . For investigating direct protein-protein interactions within the NDC80 complex, proximity ligation assays (PLA) offer in situ visualization of molecular proximities with high specificity. Alternatively, FRET (Förster Resonance Energy Transfer) techniques using fluorescently-labeled components can measure real-time interactions and determine binding affinities between SPC24 and other NDC80 complex members.

How can researchers differentiate between SPC24 expression in normal versus pathological conditions?

Distinguishing SPC24 expression between normal and pathological conditions requires multi-modal analytical approaches. Research demonstrates that SPC24 is expressed at significantly higher levels in diseased prostatic tissues (prostatitis, BPH, and cancer) compared to adjacent/normal tissues . This differential expression can be quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, and western blotting . For accurate differentiation, researchers should employ tissue microarrays with matched normal-tumor samples, analyze both protein and mRNA expression levels, and incorporate appropriate housekeeping controls. Statistical thresholds for defining "high" versus "low" expression should be established using ROC analysis based on clinically relevant endpoints .

What methodological approaches help evaluate SPC24 as a potential diagnostic biomarker?

Evaluating SPC24 as a diagnostic biomarker requires systematic validation across multiple analytical platforms. Research indicates that SPC24 may serve as a promising biomarker for prostate diseases . Methodologically, researchers should implement a multi-phase biomarker validation approach including: (1) discovery phase using high-throughput proteomics or transcriptomics; (2) verification phase using targeted assays like ELISA with biotin-conjugated antibodies; (3) validation phase with larger, independent cohorts; and (4) clinical utility assessment. For prostate cancer specifically, combining SPC24 with established markers (PSA) and related proteins (NDC80, BUB1) improves diagnostic accuracy . ROC analysis should assess sensitivity, specificity, and area under the curve (AUC) to determine diagnostic performance relative to gold standard methods.

What strategies minimize background interference when using biotin-conjugated SPC24 antibodies?

Minimizing background interference with biotin-conjugated antibodies requires addressing several technical factors. Endogenous biotin in biological samples represents a primary concern, especially in biotin-rich tissues like liver, kidney, and brain. Pre-blocking with unconjugated avidin/streptavidin effectively sequesters endogenous biotin. Additionally, including 0.02% sodium azide in buffer systems helps prevent bacterial contamination that could contribute to background . When performing ELISA, the traditional challenge of inconsistent antigen binding to plates can be overcome by using derivatization approaches (such as with adipic acid dihydrazide) prior to biotinylation . For immunohistochemical applications, incorporating appropriate blocking steps with 5-10% normal serum from the same species as the secondary reagent reduces non-specific binding.

What factors affect the stability and shelf-life of biotin-conjugated SPC24 antibodies?

Several factors influence the stability and shelf-life of biotin-conjugated antibodies. Storage conditions significantly impact longevity, with optimal preservation achieved in buffer systems containing 50% glycerol at pH 7.3 and stored at -20°C . Repeated freeze-thaw cycles accelerate degradation and should be minimized through preparation of single-use aliquots. The biotin-to-protein ratio impacts long-term stability, with over-biotinylation potentially compromising antigen recognition and increasing aggregation propensity. Inclusion of 0.02% sodium azide as a preservative prevents microbial growth, though researchers should note potential inhibitory effects on HRP in downstream applications . For maximum shelf-life, carrier proteins (e.g., BSA) may be added at 0.1-1% to prevent adsorption to container surfaces and enhance freeze-thaw stability.