SPC24 Antibody, FITC conjugated

What is SPC24 and what is its biological significance?

SPC24 functions as an essential component of the kinetochore-associated NDC80 complex, which plays a critical role in chromosome segregation and spindle checkpoint activity. It is required for maintaining kinetochore integrity and organizing stable microtubule binding sites in the outer plate of the kinetochore. The NDC80 complex, including SPC24, synergistically enhances the affinity of the SKA1 complex for microtubules and may enable the NDC80 complex to track depolymerizing microtubules during cell division . Understanding SPC24's function is crucial because disruption of both SPC24 and SPC25 genes renders cells spindle checkpoint defective, allowing them to bypass mitosis without proper chromosomal segregation, resembling early tumorigenesis events .

What is the molecular structure and characteristics of SPC24?

SPC24 (also known as SPBC24, Kinetochore protein Spc24, or hSpc24) is a 22 kDa protein involved in the kinetochore-microtubule interface . The protein contains functional domains that facilitate interaction with other NDC80 complex components. When studying SPC24, researchers should note that antibodies targeting different amino acid regions may have different specificity and applications. For example, some antibodies target AA 21-97, which is a functionally significant region of the protein . The full protein consists of 197 amino acids, with some antibodies recognizing the entire sequence (AA 1-197) while others target specific regions such as AA 1-50 or AA 21-97 .

What types of SPC24 antibodies are available for research?

Multiple types of SPC24 antibodies are available for research applications, including:

Researchers should select the appropriate antibody based on their experimental needs, including the application, target species, and preferred detection method .

What applications is the FITC-conjugated SPC24 antibody suitable for?

The FITC-conjugated SPC24 antibody is particularly well-suited for applications that benefit from direct fluorescence detection, including:

• Flow cytometry: For quantification and characterization of SPC24-expressing cells in heterogeneous populations

• Immunocytochemistry (ICC): For visualization of SPC24 localization within cells

• Immunohistochemistry (IHC): For detection of SPC24 in tissue sections, with recommended dilutions of 1:20-1:200

• Immunofluorescence microscopy: For co-localization studies with other kinetochore components

When using the FITC-conjugated antibody, researchers should be aware that FITC has an excitation maximum at approximately 495 nm and an emission maximum at around 519 nm, which should be considered when designing multi-color experiments to avoid spectral overlap .

How should researchers optimize immunofluorescence protocols for SPC24 detection?

To optimize immunofluorescence protocols for SPC24 detection using FITC-conjugated antibodies:

• Fixation method selection: Use 4% paraformaldehyde for preserving protein epitopes while maintaining cellular structure. For enhanced nuclear protein detection, methanol fixation may be preferable.

• Permeabilization optimization: Test different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% Saponin) to ensure antibody access to nuclear/kinetochore targets.

• Blocking optimization: Use 5-10% normal serum from the same species as the secondary antibody (if using indirect detection) or BSA to reduce non-specific binding.

• Antibody dilution: Start with manufacturer recommendations (e.g., 1:20-1:200 for IHC applications) and perform a dilution series to determine optimal signal-to-noise ratio .

• Counterstaining: Use DAPI or Hoechst for nuclear counterstaining to provide context for kinetochore localization.

• Antifade mounting: Use an appropriate antifade mounting medium to prevent photobleaching of the FITC fluorophore during imaging and storage.

• Imaging parameters: Calibrate exposure settings to prevent photobleaching while maximizing signal detection.

What controls should be included when using FITC-conjugated SPC24 antibody?

Rigorous experimental design requires appropriate controls when using FITC-conjugated SPC24 antibody:

Including these controls enables proper interpretation of results and troubleshooting of unexpected findings .

What are the optimal storage conditions for maintaining FITC-conjugated SPC24 antibody activity?

For maximum retention of antibody activity and fluorophore integrity:

• Store the FITC-conjugated SPC24 antibody at 4°C for up to 12 months in the supplied buffer solution containing 50% glycerol and 0.09% sodium azide .

• For longer-term storage, aliquot the antibody to avoid repeated freeze-thaw cycles, which can degrade both the antibody and the FITC conjugate.

• Protect from light at all times to prevent photobleaching of the FITC fluorophore, which is particularly sensitive to light exposure.

• Store in amber tubes or wrap containers in aluminum foil when not in use.

• Avoid exposure to extreme pH conditions or oxidizing agents that may damage the FITC conjugate.

• When removing from storage, allow the antibody to equilibrate to room temperature before opening to prevent condensation that could introduce contaminants .

How should researchers handle FITC-conjugated antibodies during experimental procedures?

To maintain optimal performance of FITC-conjugated SPC24 antibodies during experiments:

• Work in reduced light conditions when handling FITC conjugates to prevent photobleaching.

• Use proper personal protective equipment, including gloves, as the preservative ProClin in the buffer solution may be harmful .

• Ensure all dilutions are made in appropriate buffers (typically PBS with 1% BSA) to maintain antibody stability.

• Prepare working dilutions immediately before use rather than storing diluted antibody.

• Centrifuge antibody vials briefly before opening to collect liquid that may have adhered to the cap or sides.

• When performing multi-color immunofluorescence, consider the potential for spectral overlap and design experiments accordingly.

• For flow cytometry applications, include proper compensation controls when using FITC alongside other fluorophores .

How can researchers address high background when using FITC-conjugated SPC24 antibody?

High background is a common challenge with fluorescent antibodies. To reduce background when using FITC-conjugated SPC24 antibody:

• Optimize blocking: Increase blocking agent concentration (5-10% normal serum or BSA) and extend blocking time (1-2 hours at room temperature).

• Adjust antibody concentration: Titrate the antibody to determine the optimal concentration that provides specific signal with minimal background. Start with the recommended 1:20-1:200 dilution range for IHC applications .

• Modify washing steps: Implement more stringent washing with PBS-T (PBS + 0.1% Tween-20), increasing both duration and number of washes.

• Reduce autofluorescence: For tissues with high autofluorescence, pretreat with sodium borohydride (10 mg/mL for 2 minutes) or commercial autofluorescence quenchers.

• Filter buffers: Use 0.22 μm filtered buffers to remove particulates that may bind antibody non-specifically.

• Optimize fixation: Excessive fixation can increase background; optimize fixation time and conditions for your specific sample type.

• Consider alternative detection systems: If FITC background remains problematic, consider using an unconjugated primary antibody with a secondary detection system that uses a different fluorophore .

What could cause weak or absent signal when using FITC-conjugated SPC24 antibody?

When experiencing weak or absent signal with FITC-conjugated SPC24 antibody, consider these potential causes and solutions:

How can SPC24 antibodies be used to study chromosome segregation defects in cancer?

SPC24 antibodies provide valuable tools for investigating chromosome segregation defects in cancer through multiple approaches:

• Comparative expression analysis: Utilize FITC-conjugated SPC24 antibodies in flow cytometry or IHC to compare expression levels between normal and cancer tissues. Research has shown that SPC24 is overexpressed in lung adenocarcinoma and its high expression is associated with advanced tumor stages .

• Co-localization studies: Combine SPC24 antibodies with other kinetochore markers to examine potential structural or functional abnormalities in the kinetochore-microtubule interface in cancer cells.

• Functional studies: Use SPC24 antibodies to validate knockdown efficiency in siRNA experiments targeting SPC24. Studies have demonstrated that SPC24 knockdown represses cell growth and promotes apoptosis in lung cancer cell lines, suggesting its oncogenic role .

• Mitotic checkpoint analysis: Apply SPC24 antibodies in immunofluorescence to visualize kinetochore-microtubule attachments during mitosis, potentially revealing mechanisms of chromosomal instability in cancer cells.

• Biomarker development: Utilize quantitative analysis of SPC24 expression with calibrated antibody-based methods to evaluate its potential as a prognostic biomarker, as high levels of SPC24 correlate with advanced stages of lung tumors .

What are the implications of SPC24 as a potential cancer biomarker?

Research using SPC24 antibodies has revealed significant implications for SPC24 as a cancer biomarker:

• Diagnostic potential: Elevated SPC24 expression has been detected in lung adenocarcinoma samples compared to normal tissues, suggesting its utility as a diagnostic marker .

• Prognostic value: High levels of SPC24 are associated with advanced stages of lung tumors, indicating its potential as a prognostic biomarker for patient stratification .

• Correlation with clinical features: SPC24 expression correlates with important clinical characteristics and risk factors in lung cancer, including staging, survival, recurrence, and smoking status .

• Therapeutic target potential: The finding that knocking down SPC24 represses cell growth and promotes apoptosis in lung cancer cell lines suggests it may be a viable therapeutic target .

• Molecular mechanism insights: SPC24 overexpression, along with other NDC80 complex components (CDCA1/NUF2, KNTC2), has been observed in multiple cancer types including colorectal and hepatocellular carcinoma, indicating a common mechanism of kinetochore dysfunction in carcinogenesis .

For researchers investigating SPC24 as a biomarker, FITC-conjugated antibodies provide a valuable tool for flow cytometric quantification and immunohistochemical visualization in clinical samples .

How can researchers design experiments to study the relationship between SPC24 and the NDC80 complex?

To investigate the relationship between SPC24 and other NDC80 complex components:

• Co-immunoprecipitation studies: Use SPC24 antibodies to pull down the protein and associated complex members, followed by Western blot analysis to identify interactions. The NDC80 complex includes other crucial components that function together with SPC24 .

• Proximity ligation assays: Combine SPC24 antibodies with antibodies against other NDC80 complex proteins (e.g., NUF2, HEC1) to visualize and quantify protein-protein interactions in situ.

• FRET analysis: Utilize fluorescently labeled antibodies against SPC24 and other complex components to measure fluorescence resonance energy transfer, providing information about protein proximity.

• Functional domain mapping: Compare antibodies targeting different regions of SPC24 (e.g., AA 21-97 vs. AA 1-197) to determine which domains are crucial for complex formation and function .

• Knockdown studies: Combine SPC24 knockdown with immunofluorescence detection of other NDC80 components to assess complex integrity and localization in the absence of SPC24 .

• Chromosome segregation assays: Use live-cell imaging with fluorescently tagged histones in combination with fixed-cell immunofluorescence using SPC24 antibodies to correlate SPC24 function with chromosome segregation outcomes.

• Microtubule binding assays: Investigate how SPC24 contributes to the NDC80 complex's ability to enhance the affinity of the SKA1 complex for microtubules .

What are the recommended protocols for using FITC-conjugated SPC24 antibody in flow cytometry?

For optimal results in flow cytometry applications:

• Sample preparation:

  • Harvest cells in exponential growth phase

  • Fix with 2% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 in PBS for 5 minutes

  • Block with 2% BSA in PBS for 30 minutes

• Antibody staining:

  • Use FITC-conjugated SPC24 antibody at optimized concentration (starting with manufacturer recommendations)

  • Incubate for 60 minutes at room temperature in blocking buffer

  • Wash 3x with PBS

  • Resuspend cells in fresh PBS for analysis

• Controls integration:

  • Include unstained cells for autofluorescence assessment

  • Include isotype control (FITC-conjugated rabbit IgG) at the same concentration

  • Include compensation controls if performing multicolor analysis

• Cell cycle analysis integration:

  • Consider co-staining with propidium iodide or DAPI for DNA content analysis

  • This allows correlation of SPC24 expression with cell cycle phases, as SPC24 function is cell cycle-dependent

How should researchers interpret SPC24 staining patterns in different cell types?

When interpreting SPC24 staining patterns:

• Normal cells: In non-cancerous cells, SPC24 typically shows:

  • Low basal expression in interphase cells

  • Increased expression during G2 and M phases

  • Distinct punctate staining at kinetochores during mitosis

  • Little to no detection in quiescent cells

• Cancer cells: In cancer cells, particularly lung adenocarcinoma:

  • Elevated expression levels compared to normal counterparts

  • Possible aberrant localization patterns

  • More cells showing positive staining due to increased proliferation rates

  • Potentially stronger staining intensity correlating with advanced tumor stages

• Cell type variations:

  • Highly proliferative tissues (bone marrow, intestinal epithelium) may show higher baseline levels

  • Terminally differentiated cells typically show minimal expression

  • Expression patterns may vary based on tissue microenvironment and stress conditions

• Subcellular localization:

  • Primarily nuclear/kinetochore localization during mitosis

  • Diffuse cytoplasmic staining in interphase

  • Alterations in this pattern may indicate dysfunction in kinetochore assembly

What quantitative approaches can be used to analyze SPC24 expression data?

For rigorous quantitative analysis of SPC24 expression:

• Immunohistochemistry quantification:

  • H-score method: Combines staining intensity (0-3) with percentage of positive cells

  • Automated image analysis using software like ImageJ with appropriate plugins

  • Tissue microarray analysis for high-throughput screening across multiple samples

• Flow cytometry quantification:

  • Mean fluorescence intensity (MFI) measurement normalized to isotype controls

  • Percent positive cells based on appropriate gating strategies

  • Correlation with cell cycle phases when co-staining with DNA dyes

• Western blot quantification:

  • Densitometric analysis normalized to loading controls (e.g., GAPDH, β-actin)

  • Comparative expression analysis between normal and cancer tissues

  • Dose-response analysis in knockdown or overexpression experiments

• Statistical analysis approaches:

  • Correlation with clinical parameters (stage, grade, survival)

  • Kaplan-Meier survival analysis stratified by SPC24 expression levels

  • Multivariate analysis to determine independent prognostic value

These quantitative approaches enable robust assessment of SPC24's potential as a biomarker and therapeutic target in cancer research .