SPCC24B10.06 Antibody

What is SPCC24B10.06 and what is its function in cellular systems?

SPCC24B10.06 is likely the Schizosaccharomyces pombe (fission yeast) homolog of the SPC24 gene. Based on comparative genomics, it belongs to a family of cell cycle-related genes that play critical roles in kinetochore assembly and chromosome segregation during mitosis. The SPC24 protein forms part of the NDC80 kinetochore complex, which is essential for proper attachment of chromosomes to the mitotic spindle. This protein has been identified as a potential biomarker and therapeutic target in cancer research, particularly in laryngeal squamous cell carcinoma (LSCC) .

How are antibodies against SPCC24B10.06 typically generated?

Antibodies against SPCC24B10.06 are typically generated through immunization of host animals with purified recombinant protein or synthetic peptides derived from the target sequence. For polyclonal antibodies, the process generally involves repeated immunization of rabbits with highly purified antigen, followed by collection of antisera and purification of IgG through affinity chromatography . For monoclonal antibodies, hybridoma technology would be employed after mouse immunization. The resulting antibodies are then validated for specificity through various techniques including Western blotting, immunoprecipitation, and immunohistochemistry to ensure they recognize the intended target with high specificity.

What are the standard applications for SPCC24B10.06 antibodies in research?

SPCC24B10.06 antibodies can be applied in multiple research techniques, including:

• Western blotting: For detection of the protein in cell or tissue lysates (typical working dilution range: 0.1-2 μg/ml)

• ELISA: For quantitative detection (typical concentration range: 1-5 μg/ml)

• Immunohistochemistry: For visualization of protein expression in tissue sections

• Immunofluorescence: For subcellular localization studies

• Immunoprecipitation: For protein-protein interaction studies

• Chromatin immunoprecipitation: For studies of DNA-protein interactions

• Flow cytometry: For quantitative analysis at the single-cell level

What is the relationship between SPCC24B10.06 and human SPC24?

SPCC24B10.06 is the S. pombe ortholog of the human SPC24 gene. Human SPC24 has been identified as a potential diagnostic and prognostic biomarker in cancer research. Recent studies have shown that SPC24 may be associated with LSCC malignancy and represents a novel therapeutic target . The evolutionary conservation of this protein across species suggests fundamental roles in cell division and chromosome segregation, making the study of SPCC24B10.06 in model organisms relevant to understanding human disease mechanisms.

What research models are appropriate for studying SPCC24B10.06 function?

The following research models are suitable for studying SPCC24B10.06 function:

What are the optimal conditions for using SPCC24B10.06 antibodies in Western blotting?

For optimal Western blotting with SPCC24B10.06 antibodies, researchers should consider the following protocol:

• Sample preparation: Lyse cells in RIPA buffer with protease inhibitors and centrifuge at 12,000 rpm to collect the supernatant

• Protein separation: Use SDS-PAGE to separate proteins based on molecular weight

• Transfer: Transfer proteins to PVDF membrane

• Blocking: Incubate membrane with blocking solution at 37°C for 2 hours

• Primary antibody: Incubate with SPCC24B10.06 antibody at a concentration of 0.1-2 μg/ml overnight at 4°C

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 2 hours

• Detection: Use enhanced DAB solution or ECL for visualization

• Analysis: Quantify using densitometry software such as ImageJ

How can researchers validate the specificity of SPCC24B10.06 antibodies?

Validation of SPCC24B10.06 antibody specificity should involve multiple complementary approaches:

• Western blot analysis showing a single band at the expected molecular weight

• Testing on knockout or knockdown samples as negative controls

• Competitive inhibition with the immunizing peptide

• Comparison of staining patterns across multiple antibodies targeting different epitopes

• Mass spectrometry confirmation of immunoprecipitated proteins

• Cross-validation with orthogonal techniques (e.g., mRNA expression)

• Verification across multiple cell types or tissues with known expression patterns

What methodological approaches are recommended for studying SPCC24B10.06 protein interactions?

To effectively study SPCC24B10.06 protein interactions, researchers should employ these methodological approaches:

• Co-immunoprecipitation: Use SPCC24B10.06 antibodies to pull down the protein complex from cell lysates under native conditions

• Proximity ligation assay: Visualize protein-protein interactions in situ within intact cells

• Yeast two-hybrid screening: Identify novel interaction partners

• Mass spectrometry of immunoprecipitated complexes: Comprehensively identify components of multiprotein complexes

• FRET or BRET analysis: Measure real-time interactions in living cells

• BiFC (Bimolecular Fluorescence Complementation): Visualize interaction-dependent fluorescence reconstitution

• Crosslinking mass spectrometry: Map interaction interfaces at amino acid resolution

How does post-translational modification of SPCC24B10.06 affect antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of SPCC24B10.06 through several mechanisms:

• Phosphorylation at sites within or adjacent to epitopes may alter antibody binding affinity

• Epitope masking can occur when modifications change protein conformation

• Some antibodies may specifically recognize or be blocked by certain PTMs

• Researchers should select antibodies based on whether they need to detect total protein or specific modified forms

• Western blotting with phosphatase treatment can help determine if phosphorylation affects antibody binding

• Multiple antibodies targeting different epitopes should be used to obtain a complete picture of protein expression and modification

What are the considerations for using SPCC24B10.06 antibodies in chromatin immunoprecipitation studies?

For successful chromatin immunoprecipitation (ChIP) studies using SPCC24B10.06 antibodies, researchers should:

• Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

• Generate DNA fragments of appropriate size (200-500 bp) through sonication

• Use 2-5 μg of SPCC24B10.06 antibody per ChIP reaction

• Include appropriate controls (IgG control, input samples)

• Validate antibody specificity for the ChIP application specifically

• Consider ChIP-sequencing to identify genome-wide binding sites

• Analyze data using appropriate bioinformatics tools for peak calling and gene ontology analysis

• Validate findings with orthogonal techniques (e.g., ChIP-qPCR)

What protocols are recommended for immunohistochemical detection of SPCC24B10.06 in tissue samples?

For optimal immunohistochemical detection of SPCC24B10.06:

• Tissue preparation: Fix tissues appropriately (formalin-fixed, paraffin-embedded sections are common)

• Antigen retrieval: Test multiple methods (heat-induced epitope retrieval in citrate or EDTA buffers)

• Blocking: Block endogenous peroxidase activity and non-specific binding sites

• Primary antibody incubation: Use SPCC24B10.06 antibody at optimized concentration (start with 1-5 μg/ml)

• Secondary antibody: Use appropriate species-specific detection system

• Visualization: Develop with DAB or other chromogens

• Counterstaining: Use hematoxylin for nuclear visualization

• Controls: Include positive and negative controls in each experiment

• Quantification: Use digital image analysis for quantitative assessment

How can SPCC24B10.06 antibodies be effectively optimized for ELISA assays?

For optimizing ELISA assays with SPCC24B10.06 antibodies:

• Plate coating: Coat with capture antibody or recombinant antigen (1-10 μg/ml)

• Blocking: Use 1-5% BSA or non-fat milk in PBS

• Sample preparation: Prepare cell or tissue lysates under conditions that preserve the native protein

• Primary antibody: Titrate SPCC24B10.06 antibody (typical range: 1-5 μg/ml)

• Secondary antibody: Use HRP-conjugated or biotin-conjugated detection antibodies

• Standard curve: Include recombinant SPCC24B10.06 protein as a standard

• Detection system: TMB substrate for colorimetric detection

• Validation: Verify specificity with positive and negative controls

• Data analysis: Use appropriate curve-fitting methods for quantification

What approaches can researchers use to study SPCC24B10.06 localization during cell cycle progression?

To study SPCC24B10.06 localization throughout the cell cycle:

• Synchronization: Use methods like double thymidine block or nocodazole treatment to synchronize cells

• Immunofluorescence microscopy: Stain fixed cells with SPCC24B10.06 antibody

• Co-staining: Label with cell cycle markers (e.g., cyclins, phospho-histone H3)

• Live-cell imaging: Use fluorescently tagged SPCC24B10.06 for real-time dynamics

• Super-resolution microscopy: Achieve nanoscale resolution of kinetochore organization

• Flow cytometry: Combine with DNA content analysis for quantitative assessment

• Biochemical fractionation: Separate cellular compartments and assess protein distribution

• Correlative light and electron microscopy: Connect protein localization with ultrastructural features

What are the recommended approaches for quantifying SPCC24B10.06 expression in experimental samples?

For accurate quantification of SPCC24B10.06 expression:

• Western blotting: Perform with dilution series of samples and standards

• Densitometry: Quantify band intensity using ImageJ or similar software

• qRT-PCR: Measure mRNA levels with validated primers

• ELISA: Use for quantitative protein measurement in cell or tissue lysates

• Mass spectrometry: Apply targeted proteomics approaches for absolute quantification

• Flow cytometry: Quantify at the single-cell level

• Digital pathology: Use image analysis algorithms for immunohistochemistry quantification

• Normalization: Always normalize to appropriate housekeeping proteins or genes

How can SPCC24B10.06 antibodies be used in combination with genetic manipulation techniques?

Combining SPCC24B10.06 antibodies with genetic manipulation offers powerful experimental approaches:

• RNAi or CRISPR knockout validation: Confirm antibody specificity using knockdown/knockout samples

• Rescue experiments: Reintroduce wild-type or mutant SPCC24B10.06 and assess functional recovery

• Domain mapping: Create truncation or point mutants to identify functional regions

• Tagging strategies: Add epitope tags for orthogonal detection methods

• Inducible expression systems: Study consequences of controlled expression

• Site-directed mutagenesis: Investigate the impact of specific modifications on antibody recognition

• Chimeric proteins: Evaluate domain functions through domain swapping

• Imaging: Combine with fluorescent protein fusions for live-cell dynamics studies

How is SPCC24B10.06/SPC24 expression linked to cancer development and progression?

SPCC24B10.06/SPC24 has important connections to cancer development:

• SPC24 has been identified as a potential diagnostic and prognostic biomarker in laryngeal squamous cell carcinoma (LSCC)

• Research indicates that SPC24 may be associated with LSCC malignancy and represents a novel therapeutic target

• As a component of the NDC80 kinetochore complex, SPC24 dysregulation can lead to chromosomal instability and aneuploidy

• Differential expression analysis has shown SPC24 to be among the hub genes differentially expressed in LSCC compared to normal tissues

• Bioinformatic analysis using robust rank aggregation (RRA) has identified SPC24 as a key differentially expressed gene in cancer

• Survival analysis has suggested a correlation between SPC24 expression and patient outcomes

• Integration of expression data with clinical features through weighted gene correlation network analysis (WGCNA) further supports its role in cancer progression

What bioinformatics approaches are most effective for analyzing SPCC24B10.06/SPC24 in cancer genomics?

Based on current research methodologies, the following bioinformatics approaches are most effective:

• Differential expression analysis: Using robust rank aggregation (RRA) analysis to identify significant expression changes across multiple datasets

• Network analysis: Applying weighted gene correlation network analysis (WGCNA) to explore associations between SPCC24B10.06/SPC24 and clinical features

• Protein-protein interaction (PPI) mapping: Using the STRING database to construct interaction networks and identify functional modules

• Pathway enrichment: Employing DAVID database for Gene Ontology (GO) and KEGG pathway analyses to understand biological context

• Survival analysis: Utilizing Kaplan-Meier curves and Cox proportional hazards regression to identify prognostic value

• ROC curve analysis: Assessing diagnostic potential through area under the curve (AUC) calculations

• Integrative multi-omics approaches: Combining transcriptomics, proteomics, and clinical data for comprehensive analysis

How can SPCC24B10.06 antibodies contribute to understanding kinetochore assembly mechanisms?

SPCC24B10.06 antibodies provide valuable tools for investigating kinetochore assembly:

• Immunoprecipitation studies can identify novel interaction partners within the kinetochore complex

• ChIP assays can map the association of kinetochore proteins with centromeric DNA

• Immunofluorescence microscopy can visualize the temporal and spatial dynamics of kinetochore assembly

• Western blotting can monitor expression levels and post-translational modifications during mitosis

• Proximity ligation assays can detect specific protein-protein interactions within the kinetochore in situ

• Mass spectrometry of immunoprecipitated complexes can comprehensively characterize kinetochore composition

• Super-resolution microscopy with specific antibodies can reveal the nanoscale architecture of kinetochores

• FRAP (Fluorescence Recovery After Photobleaching) can assess protein turnover within the kinetochore structure

What emerging applications of SPCC24B10.06/SPC24 are developing in translational research?

Several promising translational applications are emerging:

• Diagnostic biomarker development: SPC24 has been identified as a potential diagnostic biomarker for LSCC

• Prognostic stratification: Expression levels may help identify high-risk patient subgroups

• Therapeutic target identification: Research suggests SPC24 as a novel therapeutic target for LSCC

• Drug development: Targeting the NDC80 complex function represents a potential cancer treatment strategy

• Companion diagnostics: SPC24 expression might predict response to mitotic inhibitors

• Precision medicine approaches: Expression patterns could guide personalized treatment decisions

• Immunotherapy development: Understanding the role of SPC24 in immune response to tumors

• Combination therapy strategies: Targeting SPC24 in conjunction with established therapies

How can researchers integrate SPCC24B10.06/SPC24 studies with broader chromosome segregation research?

Integration strategies for comprehensive chromosome segregation research include:

• Combining SPCC24B10.06 antibody staining with FISH to correlate protein localization with chromosomal abnormalities

• Implementing multi-color live-cell imaging to simultaneously track kinetochore components and chromosomes

• Applying ChIP-seq to map genome-wide binding patterns of kinetochore proteins

• Utilizing proteomics approaches to characterize the dynamic interactome throughout mitosis

• Developing mathematical models of kinetochore-microtubule attachments incorporating experimental data

• Correlating protein expression with aneuploidy in clinical samples

• Employing CRISPR-Cas9 genome editing to study the effects of specific mutations on chromosome segregation

• Integrating structural biology techniques (cryo-EM, X-ray crystallography) with functional studies