CENPA (Ab-7) Antibody

What is CENP-A and why is it significant in chromatin research?

CENP-A (Centromere Protein A) is a histone H3 variant specifically incorporated into centromeric nucleosomes, serving as the epigenetic mark that defines centromere identity and function. This 17 kDa protein plays a crucial role in kinetochore assembly and chromosomal segregation during cell division. CENP-A is particularly significant in chromatin research because it represents a specialized chromatin domain essential for genome stability. Understanding CENP-A incorporation, modification, and regulation provides critical insights into chromosome biology, cell cycle progression, and genetic inheritance mechanisms. Various studies have shown that CENP-A nucleosomes form distinctive rosette-like structures around centromeres, suggesting a specialized higher-order organization crucial for proper centromere function .

How do CENP-A antibodies differ in their target epitopes and applications?

CENP-A antibodies differ primarily in their target epitopes, with some recognizing total CENP-A protein while others specifically detect post-translationally modified forms, such as phosphorylated CENP-A at serine 7. This distinction is critical for experimental design and interpretation. For instance, Phospho-CENP-A (Ser7) antibodies specifically recognize CENP-A phosphorylated at serine residue 7, a modification associated with mitotic regulation . Antibodies like CENP-A (C51A7) recognize the total protein regardless of modification state . These antibodies also vary in their applications; while both types may be suitable for Western blotting (dilution 1:1000), phospho-specific antibodies often provide additional utility in immunoprecipitation (dilution 1:25) and immunofluorescence (dilution 1:100) applications when studying the dynamics of CENP-A phosphorylation during cell cycle progression .

What species cross-reactivity can be expected from CENP-A antibodies?

CENP-A antibodies show varying degrees of species cross-reactivity depending on epitope conservation and antibody design. According to the product information, Phospho-CENP-A (Ser7) antibody shows reactivity with human samples (designated as "H") but may potentially cross-react with other species that share high sequence homology at the phosphorylation site . In contrast, the CENP-A (C51A7) Rabbit mAb demonstrates specific reactivity with mouse samples (designated as "M") . When selecting a CENP-A antibody for cross-species experimentation, researchers should carefully examine both the documented reactivity and the sequence conservation in the target epitope region across species. Some antibodies may recognize protein sequences with 100% homology across multiple species, though formal validation may not be provided for all potential reactive species.

What are the optimal conditions for using CENP-A antibodies in Western blotting experiments?

For optimal Western blotting results with CENP-A antibodies, researchers should implement the following protocol:

• Sample preparation: Extract total protein from cell or tissue samples using a denaturing lysis buffer containing protease and phosphatase inhibitors (especially important when using phospho-specific antibodies).

• Gel electrophoresis: Use 12-15% SDS-PAGE gels to achieve optimal separation of the 17 kDa CENP-A protein .

• Transfer: Employ a semi-dry or wet transfer system with PVDF membrane (0.2 μm pore size) at 100V for 60 minutes in cold transfer buffer with 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute CENP-A antibodies 1:1000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use appropriate HRP-conjugated secondary antibodies (anti-rabbit IgG) followed by enhanced chemiluminescence detection.

• Analysis: Expect to observe a distinct band at approximately 17 kDa, which corresponds to the molecular weight of CENP-A .

When using phospho-specific antibodies like Phospho-CENP-A (Ser7), it is particularly important to maintain samples at cold temperatures and use phosphatase inhibitors throughout the extraction process to preserve the phosphorylation status.

How should researchers optimize immunofluorescence protocols for CENP-A detection?

For successful immunofluorescence detection of CENP-A in cellular samples:

• Cell preparation: Culture cells on glass coverslips and fix with either 4% paraformaldehyde (10 minutes at room temperature) or ice-cold methanol (5 minutes).

• Permeabilization: Permeabilize cells with 0.2% Triton X-100 in PBS for 5 minutes if using paraformaldehyde fixation (not necessary after methanol fixation).

• Blocking: Block with 1-3% BSA in PBS for 30-60 minutes at room temperature.

• Primary antibody: Dilute Phospho-CENP-A (Ser7) antibody 1:100 in blocking solution and incubate overnight at 4°C .

• Washing: Perform 3-5 washes with PBS containing 0.1% Tween-20.

• Secondary antibody: Apply fluorescently-labeled anti-rabbit secondary antibodies (1:500 dilution) for 1 hour at room temperature, protected from light.

• Counterstaining: Counterstain with DAPI (1:1000) to visualize nuclei.

• Mounting: Mount with anti-fade mounting medium and seal edges.

• Imaging: For optimal visualization of centromeric structures, use confocal microscopy with appropriate filter sets. High-resolution imaging techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) may be required to resolve individual CENP-A nucleosome clusters and their rosette-like structures .

It is important to include appropriate controls, including secondary antibody-only controls to assess background and positive controls where CENP-A expression or phosphorylation is known to be high.

What are the key considerations for CENP-A immunoprecipitation experiments?

For effective immunoprecipitation (IP) of CENP-A:

• Lysate preparation: Prepare cell lysates in non-denaturing buffer containing protease and phosphatase inhibitors. For chromatin-associated proteins like CENP-A, consider using specialized nuclear extraction protocols.

• Antibody dilution: For Phospho-CENP-A (Ser7) antibody, use a 1:25 dilution as recommended in product specifications .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Immunoprecipitation: Incubate pre-cleared lysates with diluted antibody overnight at 4°C with gentle rotation.

• Bead addition: Add protein A/G beads and incubate for 1-3 hours at 4°C.

• Washing: Perform 4-5 stringent washes with cold IP buffer to remove non-specifically bound proteins.

• Elution: Elute immunoprecipitated complexes by boiling in SDS sample buffer.

• Analysis: Analyze immunoprecipitated material by Western blotting, mass spectrometry, or other appropriate techniques.

When studying CENP-A interacting partners or chromatin complexes, consider performing chromatin immunoprecipitation (ChIP) using cross-linking agents like formaldehyde to preserve protein-DNA interactions. This approach is particularly valuable for investigating centromeric chromatin organization and the rosette-like structures formed by CENP-A nucleosomes .

How can super-resolution microscopy enhance CENP-A localization studies?

Super-resolution microscopy techniques offer significant advantages for CENP-A localization studies by overcoming the diffraction limit of conventional light microscopy:

• Structural insights: Super-resolution techniques have revealed that CENP-A nucleosomes form distinctive rosette-like clusters around centromeres, a finding not discernible with conventional microscopy . These structures undergo specific conformational changes during G1 phase that correlate with the deposition of newly synthesized CENP-A.

• Recommended techniques:

  • PALM (Photoactivated Localization Microscopy) enables visualization of individual CENP-A molecules within nucleosome clusters .

  • dSTORM (direct Stochastic Optical Reconstruction Microscopy) provides similar single-molecule resolution for CENP-A localization studies .

  • SIM (Structured Illumination Microscopy) offers a good balance between resolution enhancement and sample preparation simplicity.

• Quantitative analysis methods:

  • Voronoi-based clustering analysis can be applied to super-resolution data to quantify CENP-A cluster characteristics including size, density, and molecular content .

  • Single-particle tracking can assess CENP-A dynamics when combined with photoactivatable or photoswitchable fluorescent protein fusions.

• Sample preparation considerations:

  • Use fixation methods that preserve cellular ultrastructure (glutaraldehyde addition can improve structural preservation).

  • Optimize antibody concentrations to achieve high specificity with minimal background.

  • Consider using smaller probes (Fab fragments or nanobodies) for improved localization precision.

These advanced imaging approaches are particularly valuable for investigating how CENP-A organization changes during the cell cycle and in response to various experimental manipulations or disease states.

What are the implications of CENP-A phosphorylation dynamics for centromere research?

CENP-A phosphorylation, particularly at serine 7, represents a critical regulatory mechanism with significant implications for centromere research:

• Cell cycle regulation: Phosphorylation of CENP-A at Ser7 is cell cycle-regulated, occurring primarily during mitosis and potentially regulating kinetochore assembly and chromosome segregation.

• Kinase involvement: Several studies suggest that Aurora kinases are responsible for CENP-A Ser7 phosphorylation, offering a potential link between mitotic kinase activity and centromere function.

• Experimental approaches:

  • Time-course studies using Phospho-CENP-A (Ser7) antibodies can track phosphorylation dynamics through cell cycle phases .

  • Kinase inhibitor treatments followed by Western blotting or immunofluorescence with phospho-specific antibodies can identify responsible kinases.

  • Site-directed mutagenesis (S7A or S7E mutations) can assess the functional significance of this phosphorylation site.

• Implications for centromere identity:

  • Phosphorylation may influence CENP-A deposition pathways.

  • Modified CENP-A may recruit specific factors to the centromere.

  • The phosphorylation state may influence the structural organization of centromeric chromatin, including the formation and maintenance of the rosette-like structures observed under super-resolution microscopy .

Understanding these phosphorylation dynamics provides insights into the molecular mechanisms underlying centromere specification and function, with potential implications for chromosomal stability in both normal and disease states.

How do CENP-A antibodies contribute to research on chromosomal instability in cancer?

CENP-A antibodies serve as essential tools in investigating chromosomal instability in cancer research through several key applications:

• Aberrant CENP-A expression: Many cancers show misregulation of CENP-A expression and localization. Using CENP-A antibodies in immunohistochemistry, Western blotting, or flow cytometry enables quantification of total CENP-A levels across different cancer types and stages.

• Ectopic CENP-A incorporation: Cancer cells often display CENP-A mislocalization to non-centromeric regions, potentially contributing to neocentromere formation and genome instability. Immunofluorescence studies using CENP-A antibodies can map these aberrant incorporation patterns.

• Post-translational modifications: Phospho-specific antibodies like Phospho-CENP-A (Ser7) help evaluate whether cancer cells exhibit altered CENP-A phosphorylation patterns that might impact chromosome segregation fidelity.

• Centromere structure analysis: Super-resolution microscopy combined with CENP-A antibodies can reveal whether the rosette-like clusters of CENP-A nucleosomes are structurally altered in cancer cells, potentially linking centromere architectural changes to chromosomal instability.

• Diagnostic and prognostic applications: Quantitative analysis of CENP-A patterns using antibody-based techniques may serve as biomarkers for certain cancer types or predict clinical outcomes related to chromosomal instability.

This research direction is particularly relevant as emerging evidence suggests that targeting centromere proteins, including CENP-A, might represent a novel therapeutic avenue for cancers characterized by chromosomal instability.

What strategies can resolve non-specific binding issues with CENP-A antibodies?

When encountering non-specific binding with CENP-A antibodies, researchers should implement the following optimization strategies:

• Antibody dilution adjustment:

  • For Western blotting: Test dilutions ranging from 1:500 to 1:2000, starting with the recommended 1:1000 dilution .

  • For immunofluorescence: Begin with 1:100 dilution but try more dilute solutions if background is high.

  • For immunoprecipitation: The recommended 1:25 dilution can be adjusted based on protein abundance.

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum) at varying concentrations (1-5%).

  • Extend blocking time from 1 hour to overnight at 4°C for particularly problematic samples.

  • Add 0.1-0.3% Triton X-100 to blocking solution to reduce hydrophobic non-specific interactions.

• Washing modifications:

  • Increase wash stringency by adding higher concentrations of detergent (0.1-0.5% Tween-20 or Triton X-100).

  • Extend washing times or increase the number of wash steps.

  • Consider using high-salt washing buffers (up to 500 mM NaCl) for particularly sticky antibodies.

• Pre-absorption:

  • Pre-absorb antibodies against fixed/permeabilized cells lacking the target protein.

  • For recombinant antibodies, pre-incubate with non-specific proteins (BSA, gelatin) before application.

• Positive and negative controls:

  • Include cell lines with known CENP-A expression levels as positive controls.

  • Use CENP-A knockout or knockdown samples as negative controls where available.

  • For phospho-specific antibodies, include samples treated with phosphatase to confirm specificity.

These approaches should be systematically tested and documented to establish optimal conditions for specific experimental systems and sample types.

How can researchers verify the specificity of phospho-CENP-A antibody signals?

Verifying the specificity of phospho-CENP-A antibody signals, particularly for Phospho-CENP-A (Ser7) antibodies , requires several complementary approaches:

• Phosphatase treatment controls:

  • Treat half of your sample with lambda phosphatase before antibody incubation.

  • Loss of signal after phosphatase treatment confirms phospho-specificity.

• Blocking peptide competition:

  • Pre-incubate the antibody with excess phosphorylated peptide (containing the Ser7 phospho-epitope).

  • Pre-incubate a parallel sample with non-phosphorylated peptide.

  • Specific signal should be blocked only by the phosphorylated peptide.

• Cell cycle synchronization:

  • Since CENP-A Ser7 phosphorylation is cell cycle-regulated, compare signal intensity between synchronized populations (e.g., G1 vs. mitotic cells).

  • Signal should be enriched in mitotic cells if the antibody is truly phospho-specific.

• Kinase inhibition experiments:

  • Treat cells with inhibitors of kinases implicated in CENP-A phosphorylation (e.g., Aurora kinase inhibitors).

  • Decreased signal following kinase inhibition supports phospho-specificity.

• Genetic approaches:

  • Express wild-type CENP-A alongside a S7A mutant (cannot be phosphorylated).

  • The phospho-specific antibody should recognize only the wild-type protein.

• Western blot validation:

  • Confirm that the antibody detects a single band at the expected molecular weight (17 kDa) .

  • Verify that the signal intensity changes as expected under conditions known to affect phosphorylation status.

What are the critical factors for reproducing CENP-A chromatin immunoprecipitation (ChIP) experiments?

Successful and reproducible CENP-A ChIP experiments depend on several critical factors:

• Crosslinking optimization:

  • For histone proteins like CENP-A, use 1% formaldehyde for 10 minutes at room temperature.

  • Consider dual crosslinking with formaldehyde followed by additional crosslinkers like DSG or EGS for capturing weaker or transient interactions.

  • Quench crosslinking with 125 mM glycine for 5 minutes.

• Chromatin fragmentation:

  • Sonication parameters must be optimized for each cell type to achieve fragments of 200-500 bp.

  • Alternative approaches include enzymatic digestion with MNase, which may better preserve nucleosome structure.

  • Verify fragmentation efficiency by agarose gel electrophoresis.

• Antibody selection and validation:

  • Test multiple antibody lots for consistent performance.

  • For total CENP-A, use antibodies recognizing conserved epitopes .

  • For modified CENP-A, phospho-specific antibodies must be validated for ChIP applications .

• Centromere enrichment assessment:

  • Design qPCR primers for known centromeric sequences where possible.

  • For highly repetitive centromeres, consider using consensus primers or sequencing-based approaches.

  • Calculate enrichment relative to input and negative control regions (non-centromeric).

• Controls to include:

  • Input controls (pre-immunoprecipitation chromatin).

  • IgG or pre-immune serum negative controls.

  • ChIP for another well-established centromere marker as positive control.

  • Spike-in normalization controls for quantitative comparisons across samples.

• Data analysis considerations:

  • For next-generation sequencing analysis of CENP-A ChIP, specialized algorithms may be needed to address the repetitive nature of centromeric DNA.

  • Correlation with super-resolution microscopy data on CENP-A rosette-like structures can provide additional validation .

Careful optimization and documentation of these parameters are essential for generating reproducible ChIP data that accurately reflects CENP-A distribution across the genome.

How does anti-CENP antibody detection relate to autoimmune disease diagnosis?

Anti-centromere antibodies (ACA), which can target various centromere proteins including CENP-A, CENP-B, and CENP-C, hold significant diagnostic value in autoimmune disease:

• Disease associations:

  • Anti-centromere antibodies are found in 20-40% of patients with systemic sclerosis (SSc), particularly the limited cutaneous subtype (lcSSc) .

  • These antibodies are included in the 2013 ACR-EULAR classification criteria for SSc diagnosis .

  • ACAs can also be detected in other autoimmune conditions, including systemic lupus erythematosus (SLE), primary biliary cholangitis, rheumatoid arthritis, and Sjögren syndrome .

• Diagnostic testing methods:

  • Indirect immunofluorescence on HEp-2 cells produces a characteristic speckled nuclear pattern.

  • ELISA and line immunoassays offer more specific detection of antibodies against individual CENP proteins.

  • Multiplex bead assays allow simultaneous detection of multiple autoantibodies including anti-CENP.

• Clinical significance:

  • Presence of anti-centromere antibodies in patients with Raynaud's phenomenon may be predictive of SSc development .

  • In established SSc, anti-centromere antibodies are associated with specific clinical manifestations including digital ulcers, pulmonary hypertension, and calcinosis.

  • Different anti-CENP specificities (e.g., anti-CENP-A vs. anti-CENP-B) may have distinct clinical associations, though CENP-B is considered the main target in most patients .

• Research directions:

  • Investigating the pathogenic role of anti-CENP antibodies in tissue damage.

  • Exploring whether antibody titers correlate with disease activity or progression.

  • Determining whether targeting specific epitopes on CENP proteins has therapeutic potential.

Understanding the relationship between anti-CENP antibodies and disease manifestations continues to evolve, with important implications for personalized medicine approaches in autoimmune disorders.

What research avenues are being explored for CENP-A as a potential biomarker?

CENP-A is emerging as a promising biomarker candidate in several research contexts:

• Cancer prognosis and stratification:

  • Altered CENP-A expression levels have been associated with poor prognosis in several cancer types.

  • Investigation of CENP-A phosphorylation status using phospho-specific antibodies like Phospho-CENP-A (Ser7) may provide additional prognostic information.

  • Spatial distribution of CENP-A (as revealed by super-resolution microscopy showing rosette-like structures ) may correlate with genomic instability and treatment response.

• Aging and senescence:

  • Changes in CENP-A incorporation and centromere integrity with cellular aging may serve as biomarkers for senescence.

  • Quantitative analysis of CENP-A levels or modifications could potentially assess biological versus chronological age.

• Reproductive health:

  • CENP-A dynamics in gametes and early embryos may indicate developmental potential.

  • Aberrant CENP-A patterns could potentially serve as biomarkers for certain forms of infertility or embryonic developmental disorders.

• Methodological approaches being developed:

  • Liquid biopsy techniques to detect circulating CENP-A protein or autoantibodies.

  • Mass spectrometry-based approaches to quantify CENP-A and its post-translational modifications.

  • Image-based cytometry for high-throughput analysis of cellular CENP-A patterns.

  • Integration of CENP-A data with other molecular markers for improved predictive accuracy.

• Challenges and considerations:

  • Standardization of detection methods across laboratories.

  • Establishing normal reference ranges for different tissue and cell types.

  • Distinguishing pathological changes from normal biological variation.

These research directions highlight the potential of CENP-A as a biomarker beyond its established role in autoimmune disease diagnosis, with applications spanning oncology, aging research, and reproductive medicine.

What emerging technologies are advancing CENP-A antibody-based research?

Several cutting-edge technologies are revolutionizing CENP-A antibody-based research:

• Proximity labeling approaches:

  • BioID and TurboID fusion with CENP-A enable in-vivo identification of centromere-proximal proteins.

  • APEX2 enzyme fusion allows temporal resolution of the CENP-A interactome through controlled peroxidase activation.

  • These approaches are revealing previously unidentified factors in centromere assembly and maintenance.

• Live-cell imaging innovations:

  • Fluorescently-tagged nanobodies against CENP-A enable real-time tracking without fusion protein expression.

  • Single-molecule tracking approaches combined with CENP-A antibody fragments provide insights into dynamic behavior.

  • FRET-based sensors detect CENP-A conformational changes and protein interactions in living cells.

• Multi-omics integration:

  • Combined ChIP-seq and proteomics (ChIP-SICAP) using CENP-A antibodies identify both DNA binding sites and protein interactions simultaneously.

  • Correlation of CENP-A localization data with transcriptomics and epigenomics provides comprehensive centromere regulation models.

  • Spatial transcriptomics near CENP-A-marked centromeres reveals potential functional RNA species.

• Cryo-electron microscopy advances:

  • ImmunoEM with CENP-A antibodies is revealing the ultrastructural organization of centromeric chromatin.

  • Correlative light and electron microscopy (CLEM) connects super-resolution fluorescence patterns of CENP-A rosette-like structures with nanoscale architectural features.

• High-throughput screening applications:

  • Automated immunofluorescence with CENP-A antibodies enables screening of compound libraries for centromere modulators.

  • CRISPR screens combined with CENP-A antibody-based readouts identify novel centromere regulators.

These technological advances are rapidly expanding our understanding of centromere biology and opening new avenues for therapeutic intervention in diseases associated with centromere dysfunction.

How can computational analysis enhance CENP-A antibody-based imaging data?

Computational approaches significantly enhance the value of CENP-A antibody-based imaging data through several advanced techniques:

• Quantitative cluster analysis:

  • Voronoi-based clustering algorithms precisely quantify CENP-A distribution patterns and rosette-like structures .

  • Ripley's K-function and pair correlation analysis assess spatial distribution and organization of CENP-A molecules.

  • Machine learning classification of CENP-A cluster morphologies can identify subtle phenotypes not apparent to human observers.

• 3D reconstruction and modeling:

  • Deconvolution algorithms improve resolution of conventional microscopy images of CENP-A structures.

  • 3D rendering from z-stack acquisitions provides volumetric information about centromere organization.

  • Particle averaging techniques, borrowed from cryo-EM, can generate consensus models of CENP-A-containing structures.

• Temporal analysis in live-cell imaging:

  • Single-particle tracking algorithms measure CENP-A dynamics, residence times, and exchange rates.

  • Hidden Markov modeling identifies distinct mobility states of CENP-A molecules.

  • Correlation analysis of CENP-A movement with cell cycle markers reveals regulatory transitions.

• Multi-channel correlation:

  • Colocalization analysis quantifies spatial relationships between CENP-A and other centromere/kinetochore components.

  • Pixel-based correlation methods assess interaction probabilities between CENP-A and candidate partners.

  • Distance mapping generates proximity networks of centromere-associated proteins relative to CENP-A.

• Large-scale image informatics:

  • Deep learning approaches for automated identification and classification of CENP-A patterns across large datasets.

  • Integration of imaging data with genomic, transcriptomic, and proteomic datasets through multimodal data fusion.

  • Cloud-based platforms for collaborative analysis of CENP-A imaging data across research groups.

These computational approaches transform descriptive CENP-A imaging into quantitative datasets suitable for statistical analysis, hypothesis testing, and integration with other experimental modalities.