CHD4 Antibody

What is CHD4 and why is it important in cellular research?

CHD4 (Chromodomain Helicase DNA-binding Protein 4, also known as Mi-2β) is an ATP-dependent helicase that binds and distorts nucleosomal DNA. It functions as a major subunit of the repressive nucleosome remodeling and deacetylase (NuRD) complex that facilitates chromatin reorganization and transcriptional regulation . CHD4 has gained significant research interest due to its critical roles in:

• DNA damage response pathway, particularly in the repair of double-strand breaks

• Chromatin remodeling and gene expression regulation

• Early B cell development

• Transcriptional repression of various genes

Given these diverse functions, CHD4 antibodies are valuable tools for investigating chromatin dynamics, DNA repair mechanisms, and cellular developmental processes.

What applications are CHD4 antibodies suitable for?

Based on commercially available antibodies and research literature, CHD4 antibodies have been validated for multiple applications:

When selecting a CHD4 antibody, consider the specific application and species reactivity requirements for your experimental system .

What are the common species cross-reactivity patterns for CHD4 antibodies?

CHD4 is highly conserved across species, allowing many CHD4 antibodies to recognize the protein from multiple organisms:

When working with less common model organisms, it's advisable to verify sequence homology or conduct preliminary validation experiments before proceeding with full-scale studies .

How can I optimize CHD4 antibody performance in chromatin immunoprecipitation (ChIP) experiments?

For optimal CHD4 ChIP and ChIP-seq results, consider the following protocol recommendations:

• Sample preparation: Use 10 μg of chromatin (approximately 4 × 10^6 cells) per IP reaction

• Antibody amount: Use 10 μl of antibody per reaction

• Validation approach: Employ SimpleChIP® Enzymatic Chromatin IP Kits for consistent results

• Controls:

  • Include IgG negative controls

  • Use positive controls targeting known CHD4 binding regions

  • Consider input chromatin controls for normalization

The high molecular weight of CHD4 (~260 kDa) requires careful optimization of chromatin fragmentation methods to ensure efficient immunoprecipitation while maintaining protein integrity .

What are the key considerations when studying CHD4 in DNA damage response pathways?

When investigating CHD4's role in DNA damage response:

• Experimental design considerations:

  • Induce DNA damage using appropriate agents (PARP inhibitors, ionizing radiation, etc.)

  • Monitor CHD4 recruitment to damaged chromatin using properly validated antibodies

  • Consider the timing of CHD4 recruitment (early response)

• Key interactions to monitor:

  • CHD4 interaction with BRIT1 (MCPH1) which is critical for HR repair

  • Co-localization with DNA repair proteins (BRCA1, RPA)

  • Association with ATM and ATR in the damage response pathway

• Functional readouts:

  • HR repair efficiency using reporter assays

  • Cellular sensitivity to DNA-damaging agents (particularly PARP inhibitors)

  • Recruitment of downstream repair factors

CHD4-deficient cells show increased sensitivity to poly(ADP-ribose) polymerase inhibitor treatment, highlighting the importance of CHD4 in the early steps of HR repair .

How can I verify the specificity of CHD4 antibodies when studying NuRD complex components?

CHD3 and CHD4 share high sequence similarity and both function as components of distinct NuRD complexes, creating potential specificity challenges:

• Cross-reactivity assessment:

  • Many antibodies exhibit weak cross-reactivity between CHD3 and CHD4

  • Validate specificity using known positive and negative controls

  • Consider using epitope-tagged constructs as references

• Validation strategies:

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Use cell lines with genetic knockouts of either CHD3 or CHD4 as specificity controls

  • Include western blot analysis with antibodies targeting unique regions of each protein

• Co-immunoprecipitation analysis:

  • Verify co-precipitation of known NuRD complex components (HDAC1, RBBP7, MTA2)

  • Mass spectrometry can confirm the presence of core factors like MTA1/3, p66, RBBP4, and MBD2/3

Research has demonstrated that CHD3 and CHD4 do not coexist in the same NuRD complex, highlighting the importance of antibody specificity when studying these distinct complexes .

How should I design experiments to investigate CHD4's role in B cell development?

Based on research findings that CHD4 is essential for early B cell development but dispensable for mature B cells , consider the following experimental approach:

• Model systems selection:

  • Use conditional knockout mouse models with stage-specific Cre drivers for in vivo studies

  • Consider Mb1^cre/wt^Chd4^fl/fl^ for early B cell stages or Cd21^cre^Chd4^fl/fl^ for mature B cells

  • For ex vivo studies, use naive splenic B cells with shRNA knockdown or CRISPR/Cas9

• Key readouts:

  • B cell development markers (B220, IgM, CD25, cKit) for bone marrow analysis

  • Class switch recombination (CSR) efficiency to IgG1 or IgG3

  • Cell survival and proliferation metrics

• Stimulation conditions:

  • LPS plus IL-4 (promotes CSR to IgG1)

  • Anti-CD40 plus IL-4 (alternative T-dependent stimulation)

  • LPS alone (evaluates CSR to IgG3)

In published studies, CHD4 depletion led to severe defects in CSR, with CHD4 KO cells switching to IgG1 at only 25% of control cells, without affecting AID expression or cell survival .

What considerations are important when studying CHD4's role in chromatin accessibility and gene expression?

To effectively investigate CHD4's function in regulating chromatin and gene expression:

• Experimental models:

  • Consider tamoxifen-inducible β-cell-specific Chd4-deficient mouse models for tissue-specific studies

  • Use human cell lines with CHD4 knockdown to validate findings across species

• Essential analyses:

  • Assess chromatin landscape changes using ATAC-seq or DNase-seq

  • Evaluate gene expression alterations through RNA-seq

  • Confirm functional consequences in relevant cellular processes (e.g., insulin secretion in β-cells)

• Integration with epigenetic marks:

  • Examine H3K9me3 patterns, as CHD4 directly binds this epigenetic mark

  • Consider the relationship between CHD4 binding sites and regions of altered chromatin accessibility

Studies in β-cells have shown that loss of CHD4 impairs whole-body glucose homeostasis and islet insulin secretion, resulting from a disordered chromatin landscape and differential gene expression programs critical for normal β-cell function .

How do I design experiments to investigate potential synthetic lethality between CHD4 deficiency and PARP inhibitors?

To explore the therapeutic potential of targeting CHD4-deficient tumors with PARP inhibitors:

• Experimental setup:

  • Generate CHD4-deficient cell lines using siRNA, shRNA, or CRISPR/Cas9

  • Treat with escalating doses of PARP inhibitors

  • Include appropriate controls (scrambled siRNA, empty vector, etc.)

• Key assays:

  • Cell viability/survival assays (MTT, CellTiter-Glo, clonogenic survival)

  • DNA damage markers (γH2AX foci, 53BP1 foci)

  • HR repair efficiency measurements

• Mechanistic investigations:

  • Monitor recruitment of DNA repair proteins (BRIT1, BRCA1, RPA) at early steps of HR repair

  • Assess chromatin remodeling at sites of DNA damage

  • Evaluate the role of CHD4 chromatin remodeling activity in PARP inhibitor sensitivity

Research has demonstrated that CHD4-depleted cells exhibit increased sensitivity to PARP inhibitor treatment due to impaired recruitment of DNA repair proteins BRIT1, BRCA1, and RPA at early steps of HR repair .

How can I address common issues when detecting CHD4 by Western blotting?

CHD4 is a high molecular weight protein (~260 kDa), which presents specific technical challenges:

For Western blotting protocol optimization:

• Use 3-8% Tris-acetate gels for better resolution of high molecular weight proteins

• Consider longer SDS-PAGE running times to achieve better separation

• Use PVDF membrane with 0.45 μm pore size for efficient transfer

• Include appropriate positive controls, such as Jurkat whole cell lysate

What are the strategies to resolve contradictory results between different CHD4 antibodies?

When facing inconsistent results with different CHD4 antibodies:

• Epitope comparison:

  • Determine the specific epitopes recognized by each antibody

  • Assess whether post-translational modifications might affect epitope recognition

  • Consider whether different antibodies might detect different CHD4 isoforms

• Validation approaches:

  • Use CHD4 knockout or knockdown samples as negative controls

  • Perform epitope competition assays

  • Consider orthogonal detection methods (mass spectrometry)

• Experimental documentation:

  • Record antibody clone numbers, lot numbers, and concentrations

  • Document all experimental conditions precisely

  • Consider testing multiple antibodies in parallel under identical conditions

• Confirming specificity:

  • Perform immunoprecipitation followed by Western blot with a different antibody

  • Assess antibody performance across different cell types and experimental conditions

  • Consider recombinant expression systems with tagged CHD4 as controls

How can I optimize CHD4 antibody performance for chromatin studies across different cell types?

When adapting CHD4 chromatin studies to different cell types:

• Cell type-specific considerations:

  • Adjust chromatin preparation methods based on cell type (adherent vs. suspension)

  • Optimize fixation conditions (time, concentration) for different cell types

  • Consider nuclear extraction efficiency differences between cell types

• Cross-linking optimization:

  • Test different formaldehyde concentrations (0.5-2%)

  • Evaluate dual cross-linking approaches (DSG followed by formaldehyde)

  • Optimize cross-linking time for each cell type

• Sonication/fragmentation adjustments:

  • Determine optimal sonication conditions for each cell type

  • Verify fragment size distribution by agarose gel electrophoresis

  • Consider enzymatic fragmentation alternatives for sensitive samples

• Cell type-specific controls:

  • Include cell type-specific positive controls (known CHD4 binding sites)

  • Validate antibody specificity in each cell type

  • Consider cell type-specific expression levels when interpreting results

How can CHD4 antibodies be utilized in cancer research and potential therapeutic development?

CHD4's role in DNA repair makes it relevant for cancer research applications:

• Biomarker development:

  • Evaluate CHD4 expression levels in different cancer types

  • Assess correlation between CHD4 expression and response to DNA-damaging therapies

  • Determine if CHD4 mutation status predicts PARP inhibitor sensitivity

• Therapeutic approaches:

  • Screen for synthetic lethality between CHD4 deficiency and various therapeutics

  • Investigate CHD4 as a potential target in combination with existing DNA repair inhibitors

  • Explore CHD4's role in cancer cell resistance mechanisms

• Experimental designs:

  • Use CHD4 antibodies for tissue microarray analyses

  • Perform immunohistochemistry on patient samples to correlate CHD4 expression with outcomes

  • Develop CHD4 activity assays for drug discovery efforts

The discovery that CHD4 deficiency sensitizes cells to PARP inhibitor treatment provides a novel approach to target CHD4-deficient tumors, potentially expanding the therapeutic applications of PARP inhibitors .

What are the latest methodologies for studying CHD4's interactions with the NuRD complex components?

Advanced techniques for investigating CHD4-NuRD interactions include:

• Proximity labeling approaches:

  • BioID or TurboID fusions to CHD4 to identify proximal proteins

  • APEX2-based proximity labeling for temporal dynamics

  • Split-BioID for studying conditional interactions

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize CHD4-NuRD complex distribution

  • Live-cell imaging with fluorescently tagged components

  • FRET/FLIM assays for direct interaction analysis

• Cryo-EM and structural studies:

  • Single-particle cryo-EM analysis of purified CHD4-NuRD complexes

  • Integrative structural biology combining cryo-EM, X-ray crystallography, and crosslinking mass spectrometry

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

• Functional genomics approaches:

  • ChIP-seq to map genome-wide binding profiles of CHD4 and other NuRD components

  • CUT&RUN or CUT&Tag for improved resolution with lower cell numbers

  • Hi-ChIP to connect chromatin interactions with CHD4 binding

Research has shown that CHD3 and CHD4 form distinct NuRD complexes with potentially different functions, highlighting the importance of precise methods to distinguish between these related but separate complexes .

How can I apply CHD4 antibodies in studying epigenetic regulation during development and differentiation?

To investigate CHD4's role in developmental processes:

• Developmental model systems:

  • Time-course analysis during differentiation of stem cells

  • Stage-specific conditional knockout models

  • Tissue-specific expression and localization studies during development

• Integrated approaches:

  • Combine CHD4 ChIP-seq with histone modification profiling (H3K9me3, etc.)

  • Correlate with developmental gene expression programs (RNA-seq)

  • Assess changes in chromatin accessibility (ATAC-seq)

• Methodological considerations:

  • Use low-input ChIP protocols for limited developmental samples

  • Consider single-cell approaches for heterogeneous developmental populations

  • Implement inducible systems for temporal control of CHD4 manipulation