CHD3 Monoclonal Antibody

What is CHD3 and what role does it play in cellular function?

CHD3 is a component of the histone deacetylase NuRD (Nucleosome Remodeling Deacetylase) complex which participates in chromatin remodeling through histone deacetylation. It plays crucial roles in anchoring centrosomal pericentrin during both interphase and mitosis, and is essential for spindle organization and centrosome integrity . CHD3 is also known by alternative names including Mi-2a, Mi2-ALPHA, and ZFH. Its involvement in chromatin structure regulation makes it an important target for epigenetic research, while its cellular architecture functions position it as a key player in cell division studies.

What are the primary research applications for CHD3 monoclonal antibodies?

CHD3 monoclonal antibodies have multiple validated applications:

• Western blotting (WB): For detecting and quantifying CHD3 protein in cell and tissue lysates (recommended dilution 1:500-1:2000)

• Immunohistochemistry (IHC): For visualizing CHD3 distribution in tissue sections (recommended dilution 1:50-1:200)

• Flow cytometry (FC): For analyzing CHD3 expression at single-cell level (recommended dilution 1:20-1:50)

Additionally, CHD3 antibodies are used in viral pathogenesis research, particularly in studies examining CHD3's role in facilitating viral ribonucleoprotein (vRNP) nuclear export through interaction with the nuclear export signal 1 (NES1) of the NS2 protein in influenza A virus .

What is the recommended protocol for Western blot analysis using CHD3 monoclonal antibodies?

For optimal Western blot detection of CHD3, follow these guidelines:

When interpreting results, note that CHD3 appears at approximately 220 kDa. Due to the large size of the protein, ensure complete denaturation to avoid false negative results .

How should samples be prepared for immunohistochemistry with CHD3 antibodies?

For IHC applications:

• Use standard formalin fixation and paraffin embedding for tissue preservation

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Apply primary antibody at 1:50-1:200 dilution, optimizing based on tissue type

• Incubate overnight at 4°C for optimal signal-to-noise ratio

• Use appropriate detection system (HRP/DAB or fluorescent secondary antibodies)

CHD3 primarily shows nuclear localization in most cell types, consistent with its chromatin remodeling function. Cytoplasmic staining should be carefully evaluated as it may represent non-specific binding .

What factors affect the successful detection of CHD3 in experimental settings?

Several factors can influence successful CHD3 detection:

• Epitope accessibility: CHD3's incorporation into the NuRD complex may limit antibody access to certain epitopes

• Protein size: The large molecular weight (~220 kDa) can affect transfer efficiency in Western blots

• Fixation conditions: Overfixation can mask epitopes, particularly in IHC applications

• Nuclear localization: Distinguishing specific nuclear signal from background can be challenging

• Cross-reactivity: Potential cross-reactivity with the related CHD4 (Mi-2β) protein

To overcome these challenges, validation with multiple techniques and comparison with transcriptional data is recommended .

What controls should be included when using CHD3 monoclonal antibodies?

Comprehensive control design for CHD3 antibody experiments should include:

• Positive controls:

  • Cell lines with documented CHD3 expression (e.g., HeLa)

  • Recombinant CHD3 protein as reference standard

• Negative controls:

  • Isotype control (Mouse IgG) at equivalent concentration

  • CHD3 knockdown/knockout samples (when available)

  • Secondary antibody-only control

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Internal tissue controls for IHC applications

For rigorous validation, consider using multiple antibodies targeting different CHD3 epitopes to confirm specificity .

How can researchers troubleshoot non-specific binding with CHD3 antibodies?

When encountering non-specific binding:

• Optimize blocking conditions:

  • Test alternative blocking agents (BSA, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.3% detergent to reduce hydrophobic interactions

• Adjust antibody parameters:

  • Perform antibody titration to identify minimum effective concentration

  • Reduce incubation time or temperature

  • Add 0.1-0.5M NaCl to increase stringency

• Improve sample preparation:

  • Implement additional washing steps

  • Pre-absorb antibody with lysate from negative control samples

  • Use specific additives to reduce non-specific interactions

If issues persist, consider testing alternative CHD3 antibody clones that target different epitopes .

How should antibody storage and handling be optimized for CHD3 monoclonal antibodies?

Proper storage and handling are critical for maintaining CHD3 antibody performance:

• Store antibody at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Formulation with 50% glycerol and 0.02% sodium azide at pH 7.3 maintains stability

• When thawing, allow antibody to reach room temperature before opening to prevent condensation

• Centrifuge briefly before opening to collect solution at the bottom of the vial

• For long-term storage, avoid diluting stock antibody solution

Following these practices will help maintain antibody activity and specificity over time .

How can CHD3 antibodies be used to investigate chromatin remodeling mechanisms?

CHD3 antibodies enable sophisticated investigations into chromatin dynamics:

• Chromatin Immunoprecipitation (ChIP): Identify genomic loci where CHD3 associates with chromatin

• Co-immunoprecipitation (Co-IP): Characterize protein interaction partners within the NuRD complex

• Proximity Ligation Assay (PLA): Visualize in situ interactions between CHD3 and other chromatin regulators

• Microscopy techniques: Track CHD3 localization during cell cycle progression or in response to stimuli

For ChIP applications, optimization of chromatin shearing is particularly important due to CHD3's association with densely packed chromatin regions. Dual crosslinking protocols often improve CHD3 ChIP efficiency .

What is the relationship between CHD3 and viral infection processes?

Research has revealed important roles for CHD3 in viral pathogenesis:

• CHD3 interacts specifically with the nuclear export signal 1 (NES1) of the NS2 protein in influenza A virus

• This interaction facilitates viral ribonucleoprotein (vRNP) nuclear export

• Disruption of the CHD3-NES1 interaction significantly delays viral vRNP export and viral propagation

CHD3 monoclonal antibodies can be utilized to:

• Immunoprecipitate viral-host protein complexes

• Block functional interactions between CHD3 and viral proteins

• Visualize co-localization of CHD3 with viral components during infection

This research direction provides insights into potential host-targeted antiviral strategies .

How can researchers resolve contradictory CHD3 detection results across different experimental approaches?

When facing contradictory results, implement a systematic troubleshooting strategy:

• Validate antibody specificity:

  • Test in CHD3 knockdown/knockout systems

  • Perform peptide competition assays

  • Compare results with multiple antibodies targeting different CHD3 epitopes

• Evaluate technical variables:

  • Sample preparation differences (fixation, lysis conditions)

  • Detection method sensitivity variations

  • Antibody lot-to-lot consistency

• Consider biological factors:

  • Cell type-specific expression patterns

  • Post-translational modifications affecting epitope recognition

  • Protein complex formation masking epitopes

• Implement orthogonal validation:

  • Complement protein detection with mRNA analysis

  • Use tagged CHD3 constructs as reference standards

  • Consider alternative detection methods

A systematic investigation controlling for these variables will typically identify the source of discrepancies .

How are microfluidics technologies advancing monoclonal antibody development for targets like CHD3?

Microfluidics technologies are revolutionizing monoclonal antibody development through:

• Single-cell encapsulation: Modern platforms can encapsulate individual antibody-secreting cells into antibody-capturing hydrogels at rates up to 10^7 cells per hour

• High-throughput screening: Flow cytometry allows for rapid selection of cells producing antigen-specific antibodies

• Improved efficiency: These approaches enable screening of millions of antibody-secreting cells with high hit rates (>85% of characterized antibodies binding target)

• Accelerated timelines: The entire process from immunization to characterized monoclonal antibodies can be completed in as little as 2 weeks

These technological advances are particularly valuable for developing antibodies against challenging targets like CHD3, especially when rapid antibody development is needed .

What considerations should researchers make regarding immunogenicity when using monoclonal antibodies in translational research?

While primarily a concern for therapeutic applications, immunogenicity considerations are relevant for translational research:

• Anti-drug antibody (ADA) formation:

  • Can neutralize antibody activity (neutralizing ADAs)

  • May alter pharmacokinetics without affecting binding (non-neutralizing ADAs)

  • Can impact experimental results in animal models

• Factors affecting immunogenicity:

  • Antibody origin (murine vs. humanized vs. fully human)

  • Administration route and frequency

  • Individual host factors including HLA haplotypes

• Detection methods:

  • ELISA-based approaches for detecting anti-antibody responses

  • Functional assays to assess neutralizing capacity

These considerations are particularly important when designing long-term in vivo studies or when transitioning from research to therapeutic development .

How can researchers validate the specificity of CHD3 antibodies for critical experiments?

Thorough validation of CHD3 antibody specificity should include:

• Genetic approaches:

  • Testing in CHD3 knockout/knockdown models

  • Comparing staining patterns in cells with varying CHD3 expression levels

• Biochemical validation:

  • Western blot confirmation of a single band at expected molecular weight (~220 kDa)

  • Peptide competition assays to confirm epitope specificity

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-reactivity assessment:

  • Testing in systems expressing related proteins (especially CHD4)

  • In silico analysis of epitope conservation across related proteins

• Application-specific validation:

  • For IHC: Comparison of staining patterns with known expression profiles

  • For IP: MS confirmation of pulled-down proteins

  • For ChIP: qPCR validation of enrichment at known target sites