CHD4 Monoclonal Antibody

What is CHD4 and why is it significant in chromatin biology research?

CHD4 (Chromodomain-helicase-DNA-binding protein 4) is an ATP-dependent helicase that binds and distorts nucleosomal DNA, acting as a key component of the histone deacetylase NuRD complex involved in chromatin remodeling . It plays critical roles in transcriptional repression, DNA damage repair, and neurogenesis. CHD4's significance stems from its fundamental role in epigenetic regulation and its dysregulation in various pathological conditions, particularly cancer . Research into CHD4 provides insights into basic mechanisms of gene expression control, DNA repair pathways, and potential therapeutic targets for diseases associated with chromatin dysregulation .

What techniques can CHD4 monoclonal antibodies be reliably used for?

CHD4 monoclonal antibodies can be reliably employed in multiple research techniques including:

• Immunohistochemistry (IHC-P) with formalin-fixed paraffin-embedded tissues

• Chromatin immunoprecipitation (ChIP) for studying CHD4-DNA interactions

• Western blotting for protein expression analysis

• Immunofluorescence for subcellular localization studies

• Flow cytometry for cell population analysis

• Immunoprecipitation for protein complex isolation and characterization

Each application requires specific optimization of antibody dilution, with recommended IHC-P dilutions typically in the range of 1:50-1:200 .

How do I determine the optimal fixation conditions for CHD4 immunohistochemistry?

For optimal CHD4 detection in immunohistochemistry:

• Start with standard formalin fixation (10% neutral buffered formalin for 24-48 hours)

• Implement heat-mediated antigen retrieval using sodium citrate buffer (pH 6.0)

• Test multiple antibody dilutions (starting with the manufacturer's recommended range of 1:50-1:200)

• Include both positive controls (tissues known to express CHD4, such as breast carcinoma) and negative controls (primary antibody omission)

• If signal is weak, extend the primary antibody incubation time (overnight at 4°C)

• For specialized applications, compare different fixatives (paraformaldehyde, methanol) to determine optimal epitope preservation

Remember that CHD4 has dual localization in the nucleus and cytoplasm, with additional specific localization to centrosomes and microtubule organizing centers .

How can I effectively design experiments to study CHD4's role in DNA damage response?

To investigate CHD4's function in DNA damage response:

• Baseline characterization:

  • Assess endogenous CHD4 expression in your cell model using the monoclonal antibody in Western blot

  • Determine CHD4 subcellular localization before damage using immunofluorescence

• DNA damage induction:

  • Apply ionizing radiation (2-4 Gy doses have shown significant effects)

  • Alternatively, use chemical agents (cisplatin, etoposide, or hydroxyurea)

• Time-course experiments:

  • Examine CHD4 recruitment to damage sites at multiple timepoints (5min, 30min, 1h, 6h, 24h)

  • Co-stain with γH2AX to mark DNA damage sites

  • Document CHD4 redistribution using the monoclonal antibody in immunofluorescence

• Functional assessment:

  • Generate CHD4 knockdown using CRISPR/Cas9

  • Compare radiosensitivity using clonogenic assays between wild-type and CHD4-depleted cells

  • Assess DNA repair kinetics via comet assay and γH2AX resolution

  • Examine impact on cell cycle using flow cytometry with the CHD4 antibody

• Mechanism exploration:

  • Perform CHD4 ChIP before and after damage to identify binding sites

  • Co-immunoprecipitate with DNA repair factors (BRCA1, 53BP1, etc.)

  • Assess PARP-dependent recruitment by combining with PARP inhibitors

Include appropriate controls and statistical analysis for each experimental approach.

What are the best strategies for optimizing CHD4 chromatin immunoprecipitation (ChIP) experiments?

For optimal CHD4 ChIP experiments:

• Crosslinking optimization:

  • Test multiple formaldehyde concentrations (1-2%) and times (5-15 minutes)

  • For CHD4's interaction with nucleosomal DNA, dual crosslinking with ethylene glycol bis(succinimidyl succinate) (EGS) followed by formaldehyde can improve efficiency

• Sonication parameters:

  • Optimize sonication to achieve chromatin fragments of 200-500bp

  • Verify fragment size by agarose gel electrophoresis before proceeding

• Antibody selection and validation:

  • Use ChIP-grade monoclonal antibodies specifically validated for this application

  • Perform preliminary IP-Western to confirm antibody efficiency

  • Include isotype control antibodies to assess non-specific binding

• CHD4-specific considerations:

  • Increase salt concentration in wash buffers to reduce background

  • Add detergents (0.1% SDS, 1% Triton X-100) to improve specificity

  • For studying CHD4 at active chromatin sites, consider using sequential ChIP with histone modification antibodies

• Data analysis:

  • Design primers for both positive control regions (known CHD4 binding sites) and negative control regions

  • Normalize to input and IgG controls

  • For genome-wide analysis, consider ChIP-seq to identify global binding patterns

Remember that CHD4's association with chromatin can be dynamic and context-dependent, requiring careful experimental timing and cellular conditions.

How should I approach studying the interaction between CHD4 and the NuRD complex components using monoclonal antibodies?

To investigate CHD4-NuRD complex interactions:

• Co-immunoprecipitation (Co-IP) strategy:

  • Use CHD4 monoclonal antibody for IP followed by blotting for other NuRD components (HDAC1/2, MBD2/3, MTA1/2/3, RBBP4/7)

  • Perform reciprocal IPs with antibodies against other NuRD components

  • Include appropriate controls (IgG, input, non-NuRD proteins)

• Buffer optimization:

  • Use gentle lysis conditions to preserve protein-protein interactions (e.g., 150mM NaCl, 0.5% NP-40)

  • Test multiple detergent concentrations to maintain complex integrity

  • Include protease and phosphatase inhibitors to prevent degradation

• Size exclusion chromatography:

  • Fractionate nuclear extracts to isolate intact NuRD complex

  • Analyze fractions by Western blot using the CHD4 monoclonal antibody

  • Compare fractionation patterns under different cellular conditions

• Proximity ligation assay (PLA):

  • Utilize CHD4 monoclonal antibody in combination with antibodies against other NuRD components

  • This allows visualization of protein interactions in situ with subcellular resolution

  • Quantify interaction signals under different experimental conditions

• CRISPR-based methods:

  • Generate endogenously tagged CHD4 using CRISPR/Cas9

  • Perform pull-downs under native conditions

  • Compare complex formation with wild-type versus mutant CHD4

This multi-faceted approach provides comprehensive insights into how CHD4 functions within the NuRD complex in different cellular contexts.

How can CHD4 monoclonal antibodies be employed to assess CHD4 as a prognostic marker in cancer samples?

To evaluate CHD4 as a cancer prognostic marker:

This comprehensive approach provides robust assessment of CHD4's prognostic significance across cancer types.

What methodological approaches should be used to investigate CHD4's role in radioresistance of cancer cells?

To study CHD4's involvement in cancer radioresistance:

• Expression correlation:

  • Compare CHD4 protein levels across radioresistant and radiosensitive cell lines using the monoclonal antibody in Western blot

  • Analyze patient samples pre- and post-radiotherapy failure with IHC-P

• Functional modulation:

  • Generate stable CHD4 knockdown cell lines using CRISPR/Cas9 (50% reduction has shown significant effects)

  • Verify knockdown efficiency by Western blot with the monoclonal antibody

  • Create complementation models with wild-type or mutant CHD4

• Radiation response assessment:

  • Perform clonogenic survival assays with increasing radiation doses (2-8 Gy)

  • Measure DNA damage and repair kinetics by comet assay and γH2AX foci formation/resolution

  • Assess cell cycle distribution and checkpoint activation using flow cytometry

  • Evaluate apoptosis induction following irradiation

• Mechanistic investigations:

  • Analyze CHD4 recruitment to DNA damage sites using the monoclonal antibody in immunofluorescence

  • Assess impact on PARP-dependent pathways, as CHD4 is recruited to PARP-dependent sites of DNA damage

  • Investigate potential synergistic effects with PARP inhibitors

  • Examine cell cycle regulation, particularly the G1/S transition where CHD4 controls p53 deacetylation

• In vivo validation:

  • Develop xenograft models with CHD4 knockdown versus control cells

  • Apply fractionated radiotherapy and measure tumor growth delay

  • Analyze tumor samples by IHC-P using the CHD4 monoclonal antibody

This systematic approach has revealed that CHD4 knockdown significantly increases radiosensitivity, particularly at clinically relevant dose fractions of 2 and 4 Gy .

How do I interpret contradictory CHD4 expression data between different cancer subtypes?

When facing contradictory CHD4 expression data across cancer subtypes:

• Technical validation:

  • Verify antibody specificity through positive and negative controls

  • Compare results from multiple antibody clones targeting different CHD4 epitopes

  • Cross-validate with orthogonal methods (Western blot, qRT-PCR, proteomics)

• Contextual analysis:

  • Stratify data by molecular subtypes and driver mutations

  • Consider HPV status in HNSCC (CHD4 has different prognostic significance in HPV-positive versus HPV-negative tumors)

  • Examine potential confounding factors (treatment history, tumor stage, differentiation)

• Subcellular localization assessment:

  • Evaluate nuclear versus cytoplasmic CHD4 expression separately

  • Correlate subcellular distribution with functional outcomes

  • CHD4 functions differently based on its localization in nucleus, cytoplasm, centrosome, or cytoskeleton

• Isoform-specific analysis:

  • Determine if different cancer types express alternative CHD4 isoforms

  • Design experiments to distinguish between isoforms (isoform-specific antibodies, RT-PCR)

  • Consider post-translational modifications that might affect antibody recognition

• Functional context:

  • Relate expression differences to the biological role of CHD4 in each tissue type

  • Consider tissue-specific interaction partners and regulatory mechanisms

  • Analyze CHD4 in relation to its NuRD complex components, which may vary by cancer type

• Statistical rigor:

  • Ensure appropriate statistical power for subgroup analyses

  • Apply multiple comparison corrections when analyzing across cancer types

  • Consider meta-analysis approaches when comparing independent datasets

This comprehensive approach helps resolve apparent contradictions and provides deeper insights into the context-dependent roles of CHD4 in different cancer settings.

How should I validate the specificity of a CHD4 monoclonal antibody for my particular application?

To validate CHD4 monoclonal antibody specificity:

• Genetic validation:

  • Test the antibody in CHD4 knockdown or knockout samples generated by CRISPR/Cas9

  • Verify signal reduction proportional to the level of knockdown (e.g., 50% protein reduction should show corresponding signal reduction)

  • Include wild-type controls processed identically

• Expression system verification:

  • Express tagged CHD4 (His, FLAG, GFP) in a model system

  • Perform parallel detection with both the CHD4 monoclonal antibody and an antibody against the tag

  • Confirm signal co-localization or co-detection

• Application-specific controls:

  • For Western blot: Verify single band at the expected molecular weight (~218 kDa)

  • For IHC/IF: Include positive control tissues (e.g., breast carcinoma) and negative control tissues

  • For ChIP: Perform parallel ChIP with isotype control and analyze known target regions

• Cross-reactivity assessment:

  • Test the antibody in samples from multiple species if cross-reactivity is claimed (human, mouse, rat)

  • Evaluate potential cross-reactivity with other CHD family members (CHD3, CHD5)

  • Conduct peptide competition assays with the immunizing peptide if available

• Orthogonal method comparison:

  • Compare results with alternative antibody clones targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Verify findings with mass spectrometry when possible

This rigorous validation approach ensures reliable and reproducible results across experimental contexts.

What are the most common technical issues when working with CHD4 antibodies and how can they be resolved?

Common technical challenges with CHD4 antibodies and their solutions:

• High molecular weight protein detection problems:

  • Issue: Poor transfer efficiency of the large CHD4 protein (~218 kDa)

  • Solution: Use extended transfer times, reduce gel percentage (6-8%), implement pulsed-field or semi-dry transfer with specific buffers for high molecular weight proteins

• Nuclear protein extraction difficulties:

  • Issue: Incomplete extraction of chromatin-bound CHD4

  • Solution: Use specialized nuclear extraction buffers with high salt (300-400mM NaCl), include nuclease treatment, and extend extraction time

• Weak signal in fixed tissues:

  • Issue: Epitope masking during fixation

  • Solution: Optimize antigen retrieval (test citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) , extend primary antibody incubation (overnight at 4°C), and test signal amplification systems

• Background in immunofluorescence:

  • Issue: Non-specific binding in IF applications

  • Solution: Increase blocking time/concentration, use alternative blocking agents (BSA, serum, commercial blockers), optimize antibody dilution, and include additional wash steps

• Inconsistent ChIP results:

  • Issue: Variable chromatin immunoprecipitation efficiency

  • Solution: Optimize crosslinking conditions, ensure consistent sonication, pre-clear lysates, and use protein A/G beads pre-blocked with BSA

• Batch-to-batch variability:

  • Issue: Different antibody lots showing variable performance

  • Solution: Test each new lot against a reference sample, maintain consistent positive controls, and consider purchasing larger amounts of a single lot for long-term studies

• Species cross-reactivity limitations:

  • Issue: Antibody not working in claimed species

  • Solution: Verify sequence homology in the epitope region, test multiple antibody concentrations, and consider species-specific antibodies for critical experiments

These troubleshooting approaches address the most common technical challenges encountered when working with CHD4 monoclonal antibodies.

What controls should be included when studying CHD4 recruitment to DNA damage sites in radioresistance experiments?

Essential controls for studying CHD4 recruitment to DNA damage sites:

• Experimental controls:

  • Untreated control: Baseline CHD4 distribution before damage induction

  • Time course controls: Multiple timepoints to capture dynamic recruitment (5min, 30min, 1h, 4h, 24h)

  • Dose response: Multiple radiation doses (2Gy, 4Gy, 8Gy) to assess dose-dependent effects

  • Positive damage marker: Co-staining with γH2AX to confirm damage site formation

  • Recovery control: Allow repair time to demonstrate CHD4 dissociation from resolved damage sites

• Technical controls:

  • Antibody specificity control: CHD4 knockdown cells to verify signal specificity

  • Secondary antibody control: Samples with secondary antibody only to assess background

  • Isotype control: Non-specific IgG of the same isotype to evaluate non-specific binding

  • Blocking peptide control: Pre-incubation with immunizing peptide to confirm epitope specificity

• Biological mechanism controls:

  • PARP inhibition: Apply PARP inhibitors to block CHD4 recruitment to damage sites, as CHD4 binds to poly(ADP-ribosyl)ated proteins

  • ATM/ATR inhibition: Use kinase inhibitors to assess dependency on these damage signaling pathways

  • ZMYND8 depletion: Knockdown ZMYND8 to block CHD4 localization to acetylated damaged chromatin

  • Cell cycle synchronization: Compare recruitment in different cell cycle phases

• Validation approaches:

  • Orthogonal damage induction: Compare IR with chemical damage agents (e.g., etoposide, bleomycin)

  • Live-cell imaging: Use fluorescently tagged CHD4 to monitor real-time recruitment kinetics

  • Biochemical fractionation: Isolate chromatin fractions before and after damage to quantify CHD4 recruitment

• Functional consequence controls:

  • DNA repair assays: Measure repair efficiency in the presence/absence of CHD4

  • Clonogenic survival: Correlate CHD4 recruitment with cellular radioresistance

This comprehensive control framework ensures robust and interpretable data on CHD4's role in the DNA damage response and radioresistance.

How can I design experiments to investigate the relationship between CHD4 and p53 in regulating cell cycle progression?

To investigate CHD4-p53 regulatory interactions:

• Baseline characterization:

  • Quantify CHD4 and p53 expression levels across the cell cycle using synchronized cells

  • Perform co-immunoprecipitation with CHD4 monoclonal antibody to assess physical interaction with p53

  • Map interaction domains through truncation mutants

• Cell cycle analysis:

  • Compare cell cycle profiles between wild-type and CHD4 knockdown cells using flow cytometry

  • Focus on G1/S transition where CHD4/NuRD controls p53 deacetylation

  • Analyze p21 expression, a key p53 target gene upregulated when CHD4 is depleted

• p53 acetylation status:

  • Assess p53 acetylation levels in CHD4-depleted versus control cells

  • Use acetylation-specific antibodies (K382, K120) for Western blot analysis

  • Compare acetylation dynamics after DNA damage with and without CHD4

• Chromatin regulation:

  • Perform sequential ChIP (CHD4 followed by p53 or vice versa) at p53 target gene promoters

  • Analyze histone modifications (H3K27ac, H3K4me3, H3K9me3) at these loci

  • Map NuRD complex recruitment to p53-regulated genes

• Rescue experiments:

  • Complement CHD4 knockdown with wild-type or mutant CHD4 constructs

  • Test HDAC inhibitors to determine if the effects are dependent on deacetylase activity

  • Perform simultaneous p53 knockdown to assess dependency of phenotypes on p53

• Stress response:

  • Compare unstressed cells with those exposed to DNA damage or other p53-activating stresses

  • Analyze how CHD4-p53 interactions change under stress conditions

  • Determine if CHD4's role in radioresistance is mediated through p53 regulation

This experimental framework will elucidate how CHD4 controls cell cycle progression through p53 deacetylation and identify potential intervention points for cancer therapy.

What techniques can be used to study the dynamics of CHD4 recruitment to chromatin in live cells?

Advanced techniques for studying CHD4 chromatin dynamics in live cells:

• Fluorescent protein fusion strategies:

  • Generate endogenous CHD4-GFP/RFP knock-in using CRISPR/Cas9

  • Create stable cell lines with doxycycline-inducible fluorescent CHD4

  • Design constructs with minimal linkers to prevent interference with function

  • Validate fusion protein functionality through rescue experiments

• Live-cell imaging approaches:

  • Implement high-speed confocal microscopy for rapid dynamics

  • Use spinning disk confocal for reduced phototoxicity in long-term imaging

  • Apply lattice light-sheet microscopy for high spatiotemporal resolution

  • Employ TIRF microscopy to analyze near-membrane CHD4 populations

• Advanced dynamic analysis techniques:

  • Fluorescence Recovery After Photobleaching (FRAP) to measure CHD4 residence time on chromatin

  • Fluorescence Loss In Photobleaching (FLIP) to assess exchange rates between nuclear compartments

  • Single-particle tracking to monitor individual CHD4 molecules

  • Pair correlation analysis to detect transient interactions

• Multi-color imaging strategies:

  • Co-express fluorescently tagged histones to visualize chromatin

  • Include DNA damage markers (53BP1, γH2AX) to study recruitment to damage sites

  • Label NuRD complex partners to analyze co-recruitment dynamics

  • Use optogenetic tools to induce local DNA damage and monitor real-time recruitment

• Quantitative analysis methods:

  • Implement automated tracking algorithms for CHD4 movement

  • Apply mathematical modeling to extract kinetic parameters

  • Develop computational approaches to correlate CHD4 dynamics with chromatin states

  • Use machine learning for pattern recognition in dynamic behaviors

• Complementary biochemical validation:

  • Perform ChIP-seq at multiple timepoints to correlate with live imaging

  • Use CUT&RUN or CUT&Tag for higher resolution genomic localization

  • Validate findings with CHD4 monoclonal antibodies in fixed cells

This multi-faceted approach provides unprecedented insights into the spatiotemporal dynamics of CHD4 chromatin interactions in living cells.

How can CHD4 monoclonal antibodies be utilized to explore the potential of CHD4 as a therapeutic target in cancer?

Utilizing CHD4 monoclonal antibodies to evaluate CHD4 as a cancer therapeutic target:

• Target validation approaches:

  • Profile CHD4 expression across cancer types and normal tissues using tissue microarrays

  • Correlate expression with clinical outcomes to identify cancer types most likely to benefit from CHD4 targeting

  • Perform synthetic lethality screens to identify genetic contexts where CHD4 inhibition is most effective

• Functional assessment:

  • Use the antibody to monitor CHD4 levels after genetic or pharmacological inhibition

  • Create inducible CHD4 knockdown systems and measure effects on cancer cell viability and proliferation

  • Assess changes in DNA damage repair capacity and radiosensitivity

  • Evaluate combination effects with standard therapies (radiation, chemotherapy)

• Mechanism exploration:

  • Perform ChIP-seq before and after CHD4 depletion to identify critical target genes

  • Use RNA-seq to determine transcriptional consequences of CHD4 inhibition

  • Analyze changes in chromatin accessibility (ATAC-seq) and histone modifications

  • Study effects on cancer-relevant pathways (DNA repair, cell cycle, apoptosis)

• Drug development applications:

  • Develop cell-based screening assays using the antibody to monitor CHD4 inhibition

  • Implement high-content imaging to assess CHD4 displacement from chromatin

  • Create proximity-based assays (BRET, FRET) with labeled antibodies for drug screening

  • Use the antibody for target engagement studies of CHD4 inhibitors

• Therapeutic window assessment:

  • Compare CHD4 dependency between cancer and normal cells

  • Identify cancer-specific vulnerabilities created by CHD4 inhibition

  • Study synthetic lethal interactions with common cancer mutations

• Translational research:

  • Develop companion diagnostic approaches using the CHD4 antibody

  • Identify biomarkers that predict response to CHD4 targeting

  • Create patient-derived xenograft models to test CHD4 inhibition strategies

Evidence suggests CHD4 inhibition could be particularly effective in radioresistant HPV-negative head and neck squamous cell carcinoma, where high CHD4 expression correlates with poor survival .

What are the key considerations when selecting between different clones of CHD4 monoclonal antibodies for specific applications?

Critical factors for selecting CHD4 monoclonal antibody clones:

• Epitope location and characteristics:

  • Identify which protein domain the antibody recognizes (chromodomain, helicase domain, C-terminal region)

  • Determine if the epitope is accessible in your application (some epitopes may be masked in native conformations)

  • Consider whether post-translational modifications might affect epitope recognition

• Application-specific validation:

  • Review validation data specifically for your intended application (Western blot, IHC, ChIP)

  • Examine representative images/data from the manufacturer for your application

  • Consider antibodies specifically designated as "ChIP-grade" for chromatin studies

• Clone characteristics:

  • Evaluate isotype (IgG1, IgG2a, etc.) which may affect secondary antibody selection

  • Consider host species (mouse, rabbit) in relation to your experimental system

  • Assess purification method (protein A/G, affinity) which affects specificity and background

• Species cross-reactivity needs:

  • Verify that the clone recognizes your species of interest (human, mouse, rat)

  • Check sequence homology in the epitope region if working with uncommon species

  • Consider species-specific antibodies for critical experiments

• Format requirements:

  • Determine if you need carrier-free preparations for conjugation

  • Consider pre-conjugated antibodies for flow cytometry or imaging

  • Evaluate if BSA or azide in the storage buffer will interfere with your application

• Performance metrics:

  • Compare sensitivity (detection limit) between different clones

  • Assess specificity (background, cross-reactivity) from validation data

  • Review literature citations using specific clones for your application

• Special considerations:

  • For multiplexing, choose clones that are compatible with other antibodies in your panel

  • For native conditions, select antibodies that recognize conformational epitopes

  • For fixed samples, prefer antibodies validated in similar fixation conditions

Creating a decision matrix with these criteria helps systematically select the optimal CHD4 antibody clone for specific research needs.

How can I adapt CHD4 immunoprecipitation protocols for studying chromatin-bound versus soluble CHD4 populations?

Protocol adaptations for differentiating chromatin-bound and soluble CHD4:

• Biochemical fractionation approach:

  Soluble CHD4 extraction:

  • Lyse cells in low-salt buffer (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl₂, 0.34M sucrose, 10% glycerol, 1mM DTT, protease inhibitors)

  • Add 0.1% Triton X-100 and incubate on ice for 5 minutes

  • Centrifuge at 1,300 × g for 4 minutes at 4°C

  • Collect supernatant as cytoplasmic/nucleoplasmic fraction

  • Perform immunoprecipitation with CHD4 monoclonal antibody

  Chromatin-bound CHD4 extraction:

  • Resuspend nuclear pellet in no-salt buffer (3mM EDTA, 0.2mM EGTA, 1mM DTT, protease inhibitors)

  • Incubate on ice for 30 minutes with gentle mixing

  • Centrifuge at 1,700 × g for 4 minutes at 4°C

  • Discard supernatant (nucleoplasmic proteins)

  • Extract chromatin-bound proteins with high-salt buffer (50mM Tris-HCl pH 7.4, 300-400mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors)

  • Sonicate briefly to release chromatin-bound proteins

  • Clarify by centrifugation and perform immunoprecipitation

• Sequential immunoprecipitation strategy:

  • Extract soluble proteins first with mild detergent

  • Perform first IP with CHD4 antibody

  • Subsequently extract chromatin-bound proteins from the residual pellet

  • Conduct second IP with the same antibody

  • Compare protein partners between fractions by mass spectrometry

• In situ chromatin binding analysis:

  • Perform detergent extraction of unfixed cells on coverslips to remove soluble proteins

  • Fix remaining chromatin-bound proteins

  • Immunostain with CHD4 monoclonal antibody

  • Compare with standard fixation protocol to assess proportion of chromatin-bound CHD4

• Quantitative assessment:

  • Use Western blotting to quantify distribution between fractions

  • Include controls for fraction purity (GAPDH for cytoplasm, histone H3 for chromatin)

  • Analyze CHD4 modifications specific to each pool

• Adaptation for specific conditions:

  • Compare CHD4 distribution before and after DNA damage

  • Assess how CHD4 redistribution correlates with radiosensitivity

  • Examine changes in response to cell cycle synchronization

This fractionation approach provides crucial insights into the dynamic regulation of CHD4 between soluble and chromatin-bound states under different cellular conditions.

What methodological approaches should be used to investigate CHD4 in specialized cell types such as stem cells or primary neurons?

Specialized approaches for studying CHD4 in stem cells and primary neurons:

• Stem cell-specific considerations:

  Technical adaptations:

  • Optimize fixation to maintain stemness markers (shorter fixation times)

  • Use gentle permeabilization to preserve nuclear architecture

  • Implement low-detergent extraction for chromatin studies

  • Adjust antibody concentrations for potentially different expression levels

  Experimental design:

  • Correlate CHD4 expression/localization with pluripotency markers (Oct4, Nanog, Sox2)

  • Track CHD4 dynamics during differentiation using time-course analysis

  • Perform CHD4 ChIP-seq to identify stem cell-specific binding sites

  • Create stem cell-specific CHD4 knockdown/knockout using inducible systems

  Functional assessments:

  • Evaluate impact of CHD4 modulation on self-renewal versus differentiation

  • Analyze changes in lineage-specific gene expression after CHD4 depletion

  • Compare CHD4-NuRD complex composition between stem cells and differentiated cells

• Primary neuron adaptations:

  Technical modifications:

  • Use neuronal-optimized fixation protocols (4% PFA, 10-15 minutes)

  • Implement antigen retrieval methods specifically validated for neural tissues

  • Extend antibody incubation times (overnight at 4°C) for better penetration

  • Use detergent-free mounting media to preserve neuronal morphology

  Neuronal-specific approaches:

  • Perform subcellular localization studies (soma vs. dendrites vs. axons)

  • Correlate CHD4 expression with neuronal maturation markers

  • Investigate CHD4 in different neuronal subtypes (glutamatergic, GABAergic)

  • Examine CHD4 response to neuronal activity or depolarization

  Functional investigations:

  • Assess CHD4's role in neurogenesis using primary neural progenitors

  • Study impact on neurite outgrowth and synaptic development

  • Analyze activity-dependent gene regulation in CHD4-depleted neurons

  • Examine CHD4 in neurodevelopmental disorder models

• Common specialized techniques:

  • Low-input ChIP protocols for limited cell numbers

  • Single-cell approaches to address heterogeneity

  • Ex vivo slice cultures for maintaining tissue architecture

  • Live imaging in organoid systems

These tailored methodological approaches account for the unique characteristics of stem cells and neurons while investigating CHD4's specialized functions in these cell types.

How might single-cell approaches with CHD4 monoclonal antibodies advance our understanding of CHD4 function in heterogeneous cancer populations?

Single-cell approaches for investigating CHD4 in heterogeneous cancers:

• Single-cell protein analysis techniques:

  • Implement mass cytometry (CyTOF) with CHD4 monoclonal antibodies to quantify expression in thousands of individual cells

  • Develop multiplex immunofluorescence panels including CHD4 and cancer subtype markers

  • Apply imaging mass cytometry for spatial context of CHD4 expression in tumor sections

  • Utilize microfluidic antibody capture for single-cell Western blotting

• Integrated multi-omics approaches:

  • Combine single-cell CHD4 protein detection with scRNA-seq using CITE-seq

  • Correlate CHD4 protein levels with chromatin accessibility using scATAC-seq

  • Implement scCUT&Tag to map CHD4 binding sites in individual cells

  • Relate CHD4 levels to DNA damage repair capacity at single-cell resolution

• Tumor heterogeneity characterization:

  • Identify distinct CHD4-high and CHD4-low subpopulations within tumors

  • Correlate CHD4 expression with cancer stem cell markers

  • Map CHD4 expression across tumor spatial gradients (center vs. periphery)

  • Track how CHD4-expressing subclones evolve during treatment

• Functional readouts at single-cell level:

  • Measure radiation sensitivity in CHD4-high versus CHD4-low cells within the same tumor

  • Assess DNA repair capacity at single-cell resolution following damage

  • Analyze cell cycle distribution in relation to CHD4 expression

  • Evaluate therapy resistance markers in CHD4-stratified populations

• Clinical applications:

  • Develop CHD4-based companion diagnostics to predict radiotherapy response

  • Identify minimal residual disease based on CHD4-expressing cells

  • Track therapy-induced changes in CHD4 expression at single-cell level

  • Correlate single-cell CHD4 patterns with patient outcomes

• Technical considerations:

  • Optimize antibody concentration and signal amplification for low abundance detection

  • Implement machine learning algorithms for unbiased cell clustering

  • Develop computational approaches to integrate CHD4 data across multiple platforms

  • Address fixation and permeabilization challenges for nuclear protein detection

This single-cell perspective would transform our understanding of CHD4's role in tumor heterogeneity and therapy resistance mechanisms.

What are the most promising experimental approaches for investigating the role of CHD4 post-translational modifications in regulating its function?

Advanced approaches for studying CHD4 post-translational modifications:

• Identification of modification sites:

  • Perform mass spectrometry analysis of immunoprecipitated CHD4 to map all modifications

  • Create modification-specific antibodies for key sites (phosphorylation, acetylation, etc.)

  • Use proximity labeling (BioID, APEX) coupled with CHD4 to identify modification enzymes

  • Apply crosslinking mass spectrometry to map interactions between modified domains

• Functional significance assessment:

  • Generate site-specific mutants (phospho-mimetic, phospho-dead) through CRISPR knock-in

  • Create inducible expression systems for modified versus unmodified CHD4

  • Perform domain-swap experiments to isolate modification-dependent regions

  • Use temporal control of modifications through optogenetic or chemical-genetic approaches

• Regulatory mechanism exploration:

  • Map kinases and phosphatases regulating CHD4 through inhibitor screens

  • Investigate how DNA damage triggers CHD4 modifications

  • Assess cell cycle-dependent changes in CHD4 modification patterns

  • Study crosstalk between different types of modifications (phosphorylation influencing ubiquitination)

• Chromatin association analysis:

  • Perform ChIP-seq with modification-specific antibodies

  • Compare genomic localization of differently modified CHD4 populations

  • Analyze how modifications affect CHD4 residence time on chromatin using FRAP

  • Study impact on nucleosome remodeling activity with in vitro assays

• Complex assembly regulation:

  • Investigate how modifications affect NuRD complex formation

  • Analyze interaction with ZMYND8 and recruitment to damaged chromatin

  • Perform size exclusion chromatography to assess complex integrity

  • Use proximity ligation assays to visualize modification-dependent interactions

• Therapeutic targeting strategies:

  • Screen for compounds that modulate specific CHD4 modifications

  • Test combination approaches targeting both CHD4 and its modifying enzymes

  • Explore synthetic lethality between CHD4 modifications and cancer mutations

  • Develop modification-specific inhibitors to disrupt key CHD4 functions

This comprehensive approach would provide unprecedented insights into how post-translational modifications regulate CHD4's diverse cellular functions, potentially revealing new therapeutic opportunities.

How might emerging technologies enhance our ability to target CHD4 therapeutically in radioresistant cancers?

Emerging technologies for therapeutic targeting of CHD4 in radioresistant cancers:

• Advanced targeting modalities:

  • Develop proteolysis-targeting chimeras (PROTACs) to induce CHD4 degradation

  • Create allosteric inhibitors targeting CHD4 ATPase activity

  • Design conformation-specific antibody-based therapeutics

  • Implement RNA-based approaches (ASOs, siRNA) with cancer-specific delivery

• Structure-guided drug development:

  • Utilize cryo-EM structures of CHD4-nucleosome complexes for rational drug design

  • Apply fragment-based screening to identify binding pockets

  • Develop ATP-competitive inhibitors for the helicase domain

  • Create domain-specific inhibitors targeting chromodomain-histone interactions

• Functional screening platforms:

  • Implement CRISPR screens to identify synthetic lethal interactions with CHD4 inhibition

  • Use high-content imaging to assess effects on DNA damage response

  • Develop patient-derived organoids to test CHD4-targeting strategies

  • Create reporter systems to monitor CHD4-dependent transcriptional repression

• Precision medicine approaches:

  • Develop CHD4 expression scoring systems as predictive biomarkers

  • Create companion diagnostics using the monoclonal antibody in IHC-P

  • Stratify patients based on CHD4 levels and HPV status

  • Identify genetic modifiers that enhance dependency on CHD4

• Combination therapy strategies:

  • Test CHD4 inhibitors with fractionated radiotherapy

  • Explore synergy with PARP inhibitors, as CHD4 is recruited to PARP-dependent damage sites

  • Investigate combinations with DNA damage response inhibitors (ATM, ATR, DNA-PK)

  • Develop temporal sequencing of CHD4 inhibition and radiation

• Delivery innovations:

  • Create tumor-targeting nanoparticles for CHD4 inhibitor delivery

  • Develop radiation-activated drug release systems

  • Design antibody-drug conjugates using CHD4 monoclonal antibodies

  • Implement localized delivery approaches for head and neck cancers