CHS7 Antibody

What is CHD7 and why is it significant for research?

CHD7 (Chromohelicase/ATPase DNA-binding protein 7) is a 350 kDa member of the SNF2/RAD54 helicase family of proteins that functions as an ATP-dependent chromatin remodeling factor. It significantly influences transcription by binding to histones (specifically H3 at Lys4) and interacts with over 10,000 sites on chromatin, particularly in locations associated with gene enhancement. The protein is predominantly associated with the nervous system, found in embryonic hypothalamus (GnRH neurons), olfactory epithelium, spinal cord, and adult preoptic hypothalamus plus hippocampus. Human CHD7 spans 2997 amino acids and contains multiple functional domains including consecutive Gln-, Pro-, and Lys-rich regions (aa 151-718), two Chromo domains (aa 800-947), a helicase ATP-binding domain (aa 980-1154), and a C-terminal helicase domain (aa 1294-1464) . Its significance lies in its role in DNA replication, chromatin modification, and transcriptional regulation, making it a valuable target for neurological development research and understanding certain genetic disorders.

What are the primary applications for CHD7 antibody in research settings?

CHD7 antibodies serve multiple critical applications in research environments, with Western blotting and immunocytochemistry being the most widely documented. For Western blot analysis, the antibody can detect CHD7 in cell lysates, as demonstrated with Jurkat human acute T cell leukemia cell line, where a specific band was detected at approximately 350 kDa using sheep anti-human CHD7 antigen affinity-purified polyclonal antibody at concentrations of 0.5 μg/mL . In immunocytochemistry applications, CHD7 can be detected in fixed cells such as the HepG2 human hepatocellular carcinoma cell line, where nuclear localization is typically observed using antibody concentrations around 10 μg/mL . Additionally, CHD7 antibodies are valuable for studying organoid systems, particularly in research exploring inner ear development and hair cell differentiation, as demonstrated in human inner ear organoids where CHD7 regulates otic lineage specification . These applications collectively enable researchers to investigate CHD7's role in development, disease processes, and cellular function across various experimental models.

How should CHD7 antibody samples be properly stored and handled?

Proper storage and handling of CHD7 antibodies is critical for maintaining their efficacy and ensuring experimental reproducibility. For long-term storage, it is recommended to use a manual defrost freezer and avoid repeated freeze-thaw cycles which can degrade antibody performance. The antibody remains stable for approximately 12 months from date of receipt when stored at -20 to -70°C in its supplied form . After reconstitution, the antibody maintains stability for 1 month when stored at 2 to 8°C under sterile conditions, or for up to 6 months when stored at -20 to -70°C under sterile conditions . During experimental procedures, antibodies should be thawed gradually at refrigeration temperatures rather than at room temperature to preserve structural integrity. Aliquoting reconstituted antibodies is highly recommended to minimize freeze-thaw cycles. When working with the antibody, researchers should use sterile technique, avoid contamination, and adhere to appropriate temperature conditions for incubations (typically room temperature or 4°C depending on the application). These careful handling practices will ensure optimal antibody performance across multiple experimental applications.

How does CHD7 antibody specificity compare across different experimental models?

CHD7 antibody specificity demonstrates notable variation across experimental models, requiring careful validation for each research context. In human cell line models, sheep anti-human CHD7 antibody shows high specificity when used on Jurkat acute T cell leukemia cells, producing a distinct 350 kDa band in Western blot analysis . Similarly, in hepatocellular carcinoma HepG2 cells, the antibody exhibits clear nuclear localization pattern consistent with CHD7's known function as a chromatin remodeling factor . Cross-reactivity analysis indicates a significant degree of conservation, with human CHD7 sharing 96% amino acid identity with mouse CHD7 over amino acids 263-457, suggesting potential utility in mouse models with appropriate validation . When comparing antibody performance in organoid systems versus traditional cell cultures, researchers have noted that three-dimensional organoid structures may require optimization of antibody concentration and incubation conditions to achieve equivalent sensitivity due to penetration challenges. The data below summarizes comparative detection efficiency across experimental systems:

In advanced research applications, validation using genetic knockdown models is strongly recommended to confirm antibody specificity in novel experimental contexts .

What are the molecular mechanisms underlying CHD7 function that researchers study using these antibodies?

Researchers utilize CHD7 antibodies to elucidate several complex molecular mechanisms governing this protein's diverse functions. CHD7 operates primarily as an ATP-dependent chromatin remodeling factor that influences gene expression by binding to enhancer elements across the genome, with documented interaction at over 10,000 chromatin sites . One key mechanism under investigation involves CHD7's selective binding to histone H3 at Lysine 4, which serves as a critical regulatory point for transcriptional control. This interaction can be visualized and quantified using CHD7 antibodies in chromatin immunoprecipitation experiments. Another significant mechanism explored is CHD7's role in developmental pathways, particularly in the nervous system, where it regulates otic lineage specification and hair cell differentiation in inner ear organoids through modulation of key developmental genes . The protein contains multiple functional domains, including two Chromo (chromatin-organizer-modifier) domains (aa 800-947), a helicase ATP-binding domain (aa 980-1154), and a C-terminal helicase domain (aa 1294-1464), each contributing to distinct aspects of its function. CHD7 antibodies enable researchers to track post-translational modifications, with at least seven utilized Ser/Thr phosphorylation sites identified that may regulate protein activity. Additionally, CHD7 is known to interact with subgroup three member CHD8, forming functional complexes that can be studied through co-immunoprecipitation using specific antibodies .

How do various CHD7 antibody types perform in detecting isoform variants?

The detection capacity of CHD7 antibodies for different isoform variants represents a critical consideration for researchers investigating tissue-specific or developmentally regulated expression patterns. Based on available data, human CHD7 exhibits at least three identified isoform variants that must be considered when designing experimental approaches . The first variant contains a 12 amino acid substitution for residues 1127-2997, potentially affecting the helicase domains' function. The second is a substantially truncated 145 kDa isoform showing deletion of amino acids 573-2621, which eliminates several functional domains including the Chromo and helicase regions. The third variant contains a 15 amino acid insertion after Ser698, with potential implications for protein folding and interaction capacity . Current commercially available antibodies show variable capacities for detecting these distinct isoforms, with most antibodies designed against epitopes in the N-terminal region (such as Ala263-Gln457) demonstrating greater ability to detect multiple variants. The following data summarizes detection efficacy:

Researchers investigating tissue-specific expression patterns should employ antibodies raised against conserved regions and validate detection using known positive controls expressing specific isoforms of interest .

What are the optimal protocols for using CHD7 antibody in Western blot applications?

The optimal Western blot protocol for CHD7 detection requires careful consideration of protein size, sample preparation, and detection parameters. As CHD7 is a large 350 kDa protein, special considerations for large protein transfer are essential. Begin by preparing cell lysates in a buffer containing protease inhibitors to prevent degradation of this high molecular weight protein. For Jurkat cell lysates, approximately 30 μg of total protein per lane is recommended for clear detection . Use a low percentage (6-7%) SDS-PAGE gel or a gradient gel (4-15%) to adequately separate proteins in the high molecular weight range, with extended running times to ensure proper resolution. The transfer step is critical - utilize a wet transfer system with reduced methanol concentration (10% versus standard 20%) in the transfer buffer, and extend transfer time to 16-18 hours at low voltage (30V) at 4°C to ensure complete transfer of this large protein. When probing membranes, the optimal concentration for sheep anti-human CHD7 antigen affinity-purified polyclonal antibody is 0.5 μg/mL, followed by HRP-conjugated anti-sheep IgG secondary antibody . Use reducing conditions and Immunoblot Buffer Group 8 for optimal results as validated in the literature. For sensitive detection, employ enhanced chemiluminescence with extended exposure times of 1-5 minutes. This protocol consistently produces a specific band for CHD7 at approximately 350 kDa in appropriate cell types, though some optimization may be required for different sample types.

How should researchers optimize immunocytochemistry protocols for CHD7 detection?

Optimizing immunocytochemistry protocols for CHD7 detection requires careful attention to fixation, permeabilization, and antibody incubation parameters to achieve specific nuclear staining patterns. Begin with immersion fixation of cultured cells, as demonstrated successfully with HepG2 human hepatocellular carcinoma cell line . A 10-minute fixation with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 for 15 minutes provides optimal results for nuclear protein detection. Blocking should be performed with 5-10% normal serum from the same species as the secondary antibody (not the primary antibody species) for 1 hour at room temperature. The concentration of Sheep Anti-Human CHD7 Antigen Affinity-purified Polyclonal Antibody should be 10 μg/mL, with incubation times of 3 hours at room temperature or overnight at 4°C for maximal sensitivity . The nuclear localization of CHD7 requires thorough washing steps (3-5 washes of 5 minutes each) to reduce background and visualize specific nuclear staining patterns. For detection, NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody has been validated to produce excellent results, with DAPI counterstaining to confirm nuclear localization . Mounting media containing antifade agents is recommended to preserve fluorescence during imaging and storage. Critical controls should include primary antibody omission and ideally a CHD7 knockdown sample to confirm specificity of the observed nuclear staining pattern.

What controls are essential when validating CHD7 antibody specificity for novel applications?

Rigorous validation of CHD7 antibody specificity is essential for novel applications to ensure reliable and reproducible research findings. The primary validation control should include a CHD7 knockout or knockdown model (using siRNA or CRISPR-Cas9) to demonstrate absence or reduction of signal in Western blot, immunocytochemistry, or other detection methods. Peptide competition assays represent another critical validation approach, wherein pre-incubation of the antibody with excess immunizing peptide (the E. coli-derived recombinant human CHD7 Ala263-Gln457 fragment) should abolish specific binding . For applications in new cell types or tissues, researchers should first confirm CHD7 expression at the mRNA level using RT-PCR or RNA-seq before proceeding with antibody-based detection. When exploring cross-reactivity in non-human species, alignment analysis of the immunogen sequence (Ala263-Gln457) with the target species is necessary, noting that human CHD7 shares 96% amino acid identity with mouse CHD7 in this region . For subcellular localization studies, co-staining with established nuclear markers is recommended to confirm the expected nuclear localization pattern observed in cells like HepG2. Additionally, multiple antibodies targeting different epitopes of CHD7 should ideally be compared to build confidence in detection specificity. The following table summarizes essential controls for different applications:

Implementation of these validation strategies ensures confidence in experimental findings, particularly when exploring CHD7 function in novel biological contexts .

What are common challenges in detecting CHD7 protein and their solutions?

Researchers frequently encounter several challenges when detecting CHD7 protein due to its high molecular weight (350 kDa) and nuclear localization. One primary challenge is poor transfer efficiency in Western blotting, resulting in weak or absent bands despite adequate protein expression. This can be addressed by implementing extended transfer times (16-18 hours) at lower voltage (30V) and reduced methanol concentration in transfer buffer (10% vs. standard 20%) . Another common issue is proteolytic degradation during sample preparation, manifesting as multiple smaller bands or smears. Researchers should use fresh protease inhibitor cocktails, maintain samples at 4°C throughout processing, and avoid repeated freeze-thaw cycles of lysates. For immunocytochemistry applications, insufficient permeabilization often prevents antibody access to nuclear CHD7, which can be resolved by extending Triton X-100 permeabilization time to 15-20 minutes or using alternative permeabilization agents like 0.5% saponin . Background fluorescence in immunostaining can be minimized by extending blocking time to 2 hours, increasing blocking serum concentration to 10%, and performing additional washing steps. When working with tissue samples, antigen retrieval optimization is critical, with heat-mediated retrieval in citrate buffer (pH 6.0) typically yielding superior results for nuclear proteins. Cross-reactivity concerns can be addressed through careful antibody dilution optimization and validation in known negative controls. The table below summarizes common challenges and their solutions:

Through systematic optimization of these parameters, researchers can overcome common detection challenges for this large nuclear protein .

How can researchers address non-specific binding and background issues with CHD7 antibodies?

Non-specific binding and background issues with CHD7 antibodies can significantly impact data interpretation and should be systematically addressed through protocol optimization. The polyclonal nature of many available CHD7 antibodies, such as Sheep Anti-Human CHD7 Antigen Affinity-purified Polyclonal Antibody, can contribute to background challenges that require specific countermeasures . Begin by implementing a more stringent blocking protocol, extending blocking time to 2 hours with 5% normal serum from the secondary antibody species, supplemented with 1% BSA and 0.1% cold fish skin gelatin to reduce non-specific interactions. For Western blotting applications, increase the concentration of Tween-20 in wash buffers to 0.1% and extend washing time to 5 × 10 minutes between antibody incubations. Using validated Immunoblot Buffer Group 8 has demonstrated improved signal-to-noise ratio for CHD7 detection . For immunofluorescence applications, pre-adsorption of secondary antibodies against fixed cells of the experimental type can dramatically reduce non-specific binding. When working with tissue sections, addition of 0.1-0.3% Triton X-100 to antibody dilution buffers improves penetration while reducing non-specific membrane associations. A sequential antibody incubation approach, with primary and secondary antibodies applied separately rather than as pre-formed complexes, often yields cleaner results for nuclear proteins like CHD7. Additionally, titration experiments to determine the minimal effective concentration (typically 0.5 μg/mL for Western blotting and 10 μg/mL for immunocytochemistry) help maximize specific signal while minimizing background . For persistent background issues, switching secondary antibody types or implementing tyramide signal amplification can provide improved signal-to-noise ratios while maintaining detection sensitivity.

What considerations are important when comparing results across different CHD7 antibody clones?

When comparing results obtained using different CHD7 antibody clones or sources, researchers must consider several critical factors to ensure valid comparisons and accurate data interpretation. First, epitope differences significantly impact detection patterns, as antibodies targeting different regions of the 2997 amino acid CHD7 protein may detect distinct isoforms or post-translationally modified variants. For example, antibodies targeting the N-terminal region (aa 263-457) will detect most isoforms including the 145 kDa truncated variant, while those targeting C-terminal regions would miss this isoform entirely . Second, antibody format (polyclonal versus monoclonal) influences detection characteristics, with polyclonal antibodies typically offering higher sensitivity but potentially lower specificity compared to monoclonals. The validation status for specific applications varies between clones - documented performance in Western blotting doesn't necessarily translate to immunoprecipitation capability, for instance. When transitioning between antibody clones during a research project, side-by-side comparisons using identical samples and protocols are essential to establish correlation factors. For quantitative analyses, standard curves using recombinant protein should be generated for each antibody clone to determine relative sensitivities and dynamic ranges. Additionally, optimal working concentrations differ between clones (ranging from 0.5-10 μg/mL for various applications of the sheep polyclonal) , necessitating individual optimization rather than direct protocol transfers. Cross-reactivity profiles also vary between antibodies, particularly important when working with non-human models, given the 96% conservation between human and mouse CHD7 in some regions but potentially lower homology in others . Finally, lot-to-lot variation, particularly for polyclonal antibodies, requires documentation of lot numbers and periodic revalidation with reference standards.

How is CHD7 antibody used in chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation (ChIP) using CHD7 antibodies has emerged as a powerful approach for investigating this protein's genomic binding sites and regulatory functions. For successful CHD7 ChIP experiments, researchers should begin with extensive crosslinking (1% formaldehyde for 15 minutes) to effectively capture interactions of this large chromatin remodeling complex. Due to CHD7's function as an ATP-dependent chromatin remodeler that influences transcription through binding to over 10,000 chromatin sites, particularly at enhancer regions, ChIP protocols require careful optimization . The sheep anti-human CHD7 polyclonal antibody has been successfully employed in ChIP applications, typically at 5-10 μg per immunoprecipitation reaction, with pre-clearing steps using protein G agarose being particularly important to reduce background. Sonication conditions must be optimized to efficiently fragment chromatin containing CHD7 binding regions, typically requiring longer sonication times than standard protocols (15-20 cycles of 30 seconds on/30 seconds off) to achieve optimal fragment sizes of 200-500 bp. For ChIP-qPCR validation, primers targeting known CHD7 binding sites, particularly those near genes involved in nervous system development, provide effective positive controls. ChIP-seq analyses have revealed CHD7's preferential binding to histones (H3 at Lys4) and association with enhancer elements, with bioinformatic analyses typically showing enrichment near genes involved in developmental pathways . Successfully immunoprecipitated CHD7-associated chromatin can be analyzed for co-occurring transcription factors or histone modifications through sequential ChIP (re-ChIP) approaches to understand the complex regulatory networks involving this chromatin remodeler. This application has been particularly valuable in understanding CHD7's role in regulating otic lineage specification and hair cell differentiation in developmental contexts .

What insights have CHD7 antibodies provided in neurodevelopmental research?

CHD7 antibodies have enabled significant advancements in neurodevelopmental research, revealing crucial roles for this chromatin remodeler in multiple aspects of nervous system formation and function. Immunohistochemical studies using CHD7 antibodies have demonstrated its expression in embryonic hypothalamus (particularly in GnRH neurons), olfactory epithelium, spinal cord, and adult preoptic hypothalamus plus hippocampus, establishing its spatiotemporal pattern during critical developmental windows . In human inner ear organoid models, CHD7 immunodetection has revealed its essential function in regulating otic lineage specification and hair cell differentiation, with antibody-based visualization demonstrating nuclear localization in developing otic progenitors . CHD7 antibodies have facilitated the identification of CHD7's interaction with the SOX2 transcription factor in neural stem cells, where it cooperatively regulates genes essential for proper CNS development. Through co-immunoprecipitation studies, researchers have discovered CHD7's interaction with subgroup three member CHD8, forming functional complexes that regulate neuronal gene expression programs . Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using CHD7 antibodies has mapped this factor's genomic binding landscape during neurodevelopment, revealing preferential association with enhancer elements controlling neuronal differentiation and axon guidance. Comparative immunohistochemistry studies in normal versus CHD7-haploinsufficient models have demonstrated dosage-sensitive requirements for CHD7 in olfactory, auditory, and hippocampal development, correlating with clinical features observed in CHARGE syndrome. These antibody-enabled discoveries have transformed our understanding of chromatin-based regulation in neurodevelopment and provided mechanistic insights into neurodevelopmental disorders arising from CHD7 dysfunction.