CENPJ Antibody, HRP conjugated

What is CENPJ and why is it an important research target?

CENPJ (Centromere Protein J), also known as CPAP, LAP, or LIP1, belongs to the TCP10 family and plays critical roles in cell division and centrosome function. This protein is essential for centriole duplication and regulates microtubule nucleation from the centrosome . CENPJ acts as a microtubule plus-end tracking protein that stabilizes centriolar microtubules while inhibiting microtubule polymerization and extension from the distal ends of centrioles . It is required for centriole elongation and STIL-mediated centriole amplification . Recent research has also demonstrated CENPJ's role in regulating cilium disassembly during neurogenesis, making it particularly relevant to developmental neurobiology studies . Mutations in CENPJ are associated with microcephaly, highlighting its significance in brain development research.

What are the advantages of using HRP-conjugated CENPJ antibodies?

HRP-conjugated antibodies offer several methodological advantages for researchers studying CENPJ:

• Direct detection without secondary antibodies, simplifying experimental workflows and reducing background signal

• Enhanced sensitivity for detecting low-abundance proteins, particularly important when studying CENPJ which can have cell cycle-dependent expression

• Compatibility with multiple detection methods including ELISA, Western blotting, and immunohistochemistry

• Quantitative capability when used with appropriate substrates that produce colorimetric, fluorescent, or chemiluminescent signals

• Longer shelf-life compared to fluorescent conjugates, providing practical benefits for laboratory storage

The HRP moiety provides enzymatic amplification of the detection signal, which is particularly valuable when studying proteins that may be expressed at relatively low levels in certain cell types or developmental stages.

What applications are HRP-conjugated CENPJ antibodies suitable for?

Based on available product information, HRP-conjugated CENPJ antibodies are primarily optimized for the following applications:

For ELISA applications specifically, HRP-conjugated CENPJ antibodies provide high sensitivity with detection capabilities ranging from concentrated to approximately 729x dilution of CENPJ overexpression lysate, making them ideal for quantitative protein detection . When using these antibodies, it is critical to optimize dilutions for each specific application and experimental system.

What controls should be included when using CENPJ antibodies?

When designing experiments with CENPJ antibodies, proper controls are essential for result validation:

• Positive control: Include known CENPJ-expressing samples such as Jurkat cells or mouse testis tissue, which have been validated to express detectable levels of CENPJ

• Negative control: Use samples with CENPJ knockdown/knockout or tissues known not to express CENPJ

• Isotype control: Include a non-specific antibody of the same isotype (rabbit IgG for most CENPJ polyclonal antibodies) to assess non-specific binding

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity

• Cross-reactivity assessment: If working with non-human samples, verify species reactivity (most commercial CENPJ antibodies react with human, mouse, and rat samples)

Each experimental design should include appropriate controls based on the specific research question and detection method employed.

How should researchers optimize dilution ratios for different applications?

Optimizing antibody dilutions is critical for obtaining specific signals while minimizing background. For CENPJ antibodies, recommended starting dilutions vary by application:

For optimal results, perform a titration experiment using a dilution series with 2-3 fold increments. For Western blots, the goal is a clear band at the expected molecular weight (153 kDa for full-length CENPJ) with minimal background . For immunofluorescence, the antibody should produce specific centrosomal staining with minimal cytoplasmic background. If working with the HRP-conjugated variant specifically, ensure the substrate concentration is standardized across all dilution tests to accurately compare signal intensities .

What are the recommended storage conditions for maintaining CENPJ antibody activity?

Proper storage is essential for preserving antibody functionality. CENPJ antibodies have specific storage requirements:

For HRP-conjugated antibodies specifically, avoid exposure to light and oxidizing agents that could compromise the enzymatic activity of the conjugate .

How can researchers troubleshoot non-specific binding with CENPJ antibodies?

Non-specific binding is a common challenge when working with antibodies against centrosomal proteins like CENPJ. Several methodological approaches can help minimize this issue:

• Optimize blocking conditions: Extend blocking time to 2 hours at room temperature using 5% BSA in TBST rather than standard milk-based blockers

• Increase washing stringency: Add an additional wash step with higher salt concentration (up to 500 mM NaCl) to reduce non-specific ionic interactions

• Pre-adsorb the antibody: Incubate with negative control lysate before application to the experimental sample

• Titrate antibody concentration: Non-specific binding often increases with excessive antibody concentrations; a dilution series can determine the optimal concentration

• Cross-linking fixation comparison: Test both paraformaldehyde and methanol fixation methods, as CENPJ epitope accessibility can vary with different fixatives

If centrosome-unrelated staining persists, validation using siRNA knockdown of CENPJ can help distinguish between specific and non-specific signals. Published literature reports successful use of CENPJ antibodies in various applications with proper optimization .

What methodologies are recommended for co-localization studies with CENPJ and other centrosomal markers?

For researchers investigating CENPJ's interactions with other centrosomal proteins, sophisticated co-localization studies are essential. Recommended methodological approaches include:

• Sequential immunostaining: When using multiple rabbit-derived antibodies (common for centrosomal proteins), implement sequential immunostaining with complete stripping between rounds using glycine buffer (pH 2.5) followed by re-blocking

• Super-resolution microscopy: Standard confocal microscopy often lacks the resolution for precise centrosomal co-localization. Techniques such as STED, SIM, or STORM provide superior resolution for centrosomal proteins

• Antibody combinations: Validated antibody pairs for co-localization studies with CENPJ include:

  • CENPJ with γ-Tubulin (1:5000, T3559; Sigma-Aldrich)

  • CENPJ with Pericentrin (1:300, 611814; BD Biosciences)

  • CENPJ with Acetylated Tubulin (1:30000, T7451; Sigma-Aldrich) for cilia studies

• Z-stack acquisition: Collect complete z-stacks through the entire centrosome (0.2μm steps) followed by deconvolution and 3D reconstruction for accurate spatial relationships

For proper analysis, implement Pearson's correlation coefficient or Manders' overlap coefficient calculations to quantify co-localization rather than relying on visual inspection alone. The centrosome's small size makes precise co-localization particularly challenging and requires rigorous quantitative approaches.

How can researchers study CENPJ's dynamic behavior during cell cycle progression?

Investigating CENPJ's dynamics throughout the cell cycle requires specialized methodological approaches:

• Cell synchronization protocols:

  • G1/S phase: Double thymidine block

  • M phase: Nocodazole treatment followed by mitotic shake-off

  • S phase: Aphidicolin treatment

• Temporal analysis: Fix cells at specific time points after synchronization release (typically 0, 2, 4, 6, 8, 10, and 12 hours) and stain for CENPJ along with cell cycle markers

• Quantitative image analysis: Measure CENPJ signal intensity at centrosomes across different cell cycle stages using automated image analysis software

• Live-cell imaging: For dynamic studies, combine with cell cycle markers such as PCNA-RFP (S phase) or Histone H2B-GFP (mitosis)

• Cell cycle marker co-staining: Pair CENPJ antibody with:

  • Cyclin E (G1/S transition)

  • PCNA or BrdU incorporation (S phase)

  • Phospho-Histone H3 (mitosis)

When analyzing results, researchers should account for the dramatic changes in centrosome structure and number throughout the cell cycle. CENPJ localization patterns shift from a focused centrosomal signal in G1 to more dispersed signals during centrosome duplication in S phase, followed by bipolar centrosomal localization during mitosis .

What are the key considerations when studying CENPJ mutations associated with microcephaly?

CENPJ mutations (associated with MCPH6 primary microcephaly) require specific experimental approaches for functional characterization:

• Patient-derived cell models:

  • Fibroblasts from patients with CENPJ mutations

  • Lymphoblastoid cell lines transformed from patient blood samples

  • iPSC-derived neural progenitors to study neurogenesis defects

• CRISPR/Cas9 genome editing: Introduction of specific patient mutations into control cell lines or model organisms for direct comparison

• Phenotypic analysis:

  • Centrosome number/morphology assessment

  • Spindle formation evaluation during mitosis

  • Cell cycle progression analysis

  • Cilia formation and disassembly dynamics

• Rescue experiments:

  • Complementation with wild-type CENPJ

  • Domain-specific mutant complementation to identify functional regions

• Brain organoid models: For studying developmental consequences in a 3D tissue-like context

When interpreting results, differentiate between direct effects of CENPJ dysfunction versus secondary consequences of cellular stress responses. The use of isogenic control lines is critical to minimize genetic background effects that might confound the interpretation of observed phenotypes .

How should researchers interpret divergent results from different CENPJ antibodies?

When faced with contradictory results using different CENPJ antibodies, systematic analysis is required:

• Epitope mapping comparison: Different antibodies target distinct regions of CENPJ. Compare the epitope regions:

  • N-terminal antibodies: May detect specific isoforms

  • Middle region antibodies: Often detect most isoforms but may have accessibility issues

  • C-terminal antibodies: Commonly used but miss truncated variants

• Validation hierarchy implementation:

  • Genetic approaches: CENPJ knockout/knockdown controls

  • Multiple antibody confirmation: Use at least two antibodies targeting different epitopes

  • Recombinant expression: Tagged CENPJ expression for antibody validation

• Technical validation:

  • Western blot confirmation of specificity at the expected 153 kDa molecular weight

  • Peptide competition assays to confirm specificity

  • Immunoprecipitation followed by mass spectrometry

• Application-specific considerations:

  • Fixation method effects: Some epitopes are sensitive to specific fixatives

  • Denaturation sensitivity: Native vs. denatured protein recognition varies between antibodies

When reporting findings, researchers should clearly specify which antibody was used (including catalog number and lot), the detailed methods employed, and acknowledge limitations of their interpretations based on the specific antibody characteristics .

What methodological approaches are recommended for studying CENPJ's role in cilia regulation?

Recent research has demonstrated CENPJ's critical function in regulating cilium disassembly, particularly in neural progenitor cells . When investigating this role, researchers should consider:

• Tissue-specific analysis methods:

  • Fixed brain tissue immunostaining for cilia markers and CENPJ

  • Primary neuronal cultures for dynamic studies

  • Neural progenitor isolation from developing cortex

• Ciliary markers for co-labeling:

  • Acetylated Tubulin (1:30000, T7451; Sigma-Aldrich) for ciliary axoneme

  • ARL13B for ciliary membrane

  • γ-Tubulin (1:5000, T3559/T5326; Sigma-Aldrich) for basal body

• Quantitative analysis parameters:

  • Cilia length measurement

  • Cilia frequency (percentage of ciliated cells)

  • Cilia disassembly rate following stimulation

• Live imaging approaches:

  • Ciliary marker fusion proteins

  • Photoswitchable fluorescent protein tagging

  • FRAP (Fluorescence Recovery After Photobleaching) for dynamics

When interpreting results, it's important to distinguish between direct effects on cilia versus indirect effects through centrosome dysfunction. The timing of analysis is particularly critical, as CENPJ-related ciliary phenotypes may be transient and cell-cycle dependent .

How can researchers accurately quantify CENPJ protein levels across different experimental conditions?

Precise quantification of CENPJ protein levels requires specialized approaches due to its relatively low abundance and complex regulation:

• Quantitative Western blot methodology:

  • Use infrared fluorescent secondary antibodies rather than traditional ECL

  • Include recombinant protein standards for absolute quantification

  • Apply GAPDH or β-actin normalization with caution due to potential cell cycle effects

• ELISA-based approaches:

  • Sandwich ELISA using matched antibody pairs for detection and capture

  • Detection sensitivity validated across a wide dilution range (729x to 3x) of CENPJ overexpression lysate

  • Standard curve generation using recombinant CENPJ protein

• Internal control selection:

  • Other centrosomal proteins with stable expression (e.g., Centrin)

  • Cell-cycle independent housekeeping genes for transcript analysis

• Mass spectrometry-based quantification:

  • Selected reaction monitoring (SRM) for absolute quantification

  • SILAC labeling for comparative studies

  • TMT labeling for multiplexed comparisons

For accurate analysis, researchers should account for CENPJ's cell-cycle dependent expression and potential post-translational modifications that may affect antibody recognition. When reporting quantitative results, include details on normalization methods, statistical approaches, and technical limitations .

What are the considerations for using CENPJ antibodies in different model organisms?

While most commercial CENPJ antibodies are developed against human protein, research often extends to model organisms. Important considerations include:

When working with non-human models:

• Epitope sequence verification: Compare the antibody's target sequence with the corresponding region in your model organism

• Validation requirements:

  • Western blot confirmation at the appropriate molecular weight

  • Positive control from species-matched tissue known to express CENPJ

  • Genetic approaches (morpholino, CRISPR) to confirm specificity

• Fixation optimization:

  • Species-specific fixation protocols may be required

  • Antigen retrieval methods often need adjustment

• Developmental timing considerations:

  • Expression patterns vary across developmental stages

  • Centrosome structure differs between species and cell types

Researchers should conduct thorough validation when using antibodies in non-human systems, even when manufacturers claim cross-reactivity .

What emerging technologies might enhance CENPJ antibody-based research?

Several cutting-edge technologies show promise for advancing CENPJ research beyond traditional antibody applications:

• Proximity labeling methods:

  • BioID fusion with CENPJ to identify proximal interacting proteins

  • APEX2 for temporally controlled labeling during specific cell cycle stages

  • Split-BioID for studying conditional interactions

• Super-resolution microscopy advancements:

  • MINFLUX for nanometer precision localization

  • Expansion microscopy to physically enlarge centrosomal structures

  • Lattice light-sheet microscopy for rapid 3D imaging with reduced phototoxicity

• Engineered antibody fragments:

  • nanobodies for live-cell imaging of CENPJ

  • scFv (single-chain variable fragments) for improved penetration

  • intrabodies for tracking endogenous CENPJ in living cells

• Multiplexed detection systems:

  • Cyclic immunofluorescence for analyzing multiple centrosomal markers

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • DNA-barcoded antibodies for highly multiplexed imaging

These emerging approaches offer potential solutions to longstanding challenges in centrosome biology research, including the small size of centrosomal structures, transient protein interactions, and the complexity of the pericentriolar material where CENPJ functions .

How can researchers integrate CENPJ antibody-based detection with functional genomics approaches?

Combining traditional antibody-based research with modern functional genomics creates powerful experimental paradigms:

• CRISPR screening approaches:

  • Genome-wide screens for modulators of CENPJ localization

  • Domain-focused mutagenesis to map functional regions

  • CRISPRi/CRISPRa for controlled expression modulation

• Transcriptomics integration:

  • Correlating CENPJ protein levels with transcriptional consequences

  • Single-cell RNA-seq to identify cell populations with differential CENPJ activity

  • Spatial transcriptomics to map CENPJ-related gene expression in tissues

• Proteomics connections:

  • Antibody-based pulldowns followed by mass spectrometry

  • Thermal proteome profiling to identify CENPJ-dependent complexes

  • Cross-linking mass spectrometry for structural interaction mapping

• Multi-omics data integration:

  • Correlation of CENPJ protein levels with phosphoproteome changes

  • Integration with chromosome conformation capture data

  • Systems biology modeling of centrosome assembly/disassembly

These integrated approaches allow researchers to move beyond descriptive studies of CENPJ localization toward mechanistic understanding of CENPJ's functional networks, regulatory mechanisms, and roles in development and disease states .