GATAD2A Antibody

What is GATAD2A and why is it significant in research?

GATAD2A (GATA Zinc Finger Domain Containing 2A) is a crucial subunit of the nucleosome remodeling and deacetylase (NuRD) complex. This complex plays a vital role in regulating gene expression through histone deacetylation and ATP-dependent chromatin remodeling activities . GATAD2A is particularly significant in research because it has been implicated in neural development and its variants have been linked to neurodevelopmental disorders (NDDs), which are classified as "NuRDopathies" . The protein contains distinct proline-rich PPPLφ motifs that facilitate interactions with MYND domains in proteins such as ZMYND8, differentiating it from its paralog GATAD2B . Additionally, GATAD2A demonstrates high expression during early neural development and plays a non-redundant role in early stem cell differentiation, making it a valuable target for developmental biology research .

What are the primary applications for GATAD2A antibodies in research?

GATAD2A antibodies are versatile research tools with multiple validated applications:

These applications allow researchers to investigate GATAD2A's expression, interactions with other proteins in the NuRD complex, and its binding to specific genomic regions to regulate gene expression .

What sample types are most suitable for GATAD2A antibody detection?

GATAD2A antibodies have demonstrated high sensitivity for detecting endogenous protein in various cell and tissue types. Based on the reactivity information, human cell lines are consistently suitable for studies using these antibodies . For antibody #17705, mouse, rat, and monkey samples also show reactivity, making this antibody more versatile for comparative studies across species . When designing experiments, researchers should consider that GATAD2A is predominantly expressed in neural tissues during development , though it is also present in other cell types. For optimal results, sample preparation should include appropriate lysis buffers that can effectively extract nuclear proteins while preserving their native conformation.

How can I distinguish between GATAD2A isoforms when using antibodies?

GATAD2A exists in multiple isoforms that appear as bands at approximately 70 and 73 kDa in Western blots . To differentiate between these isoforms, consider the following approach:

• Select an antibody that recognizes an epitope common to all isoforms of interest. For example, the E7B5B rabbit monoclonal antibody (#17705) is produced against a synthetic peptide corresponding to residues surrounding Pro627 of human GATAD2A protein .

• Use high-resolution SDS-PAGE with extended running times to achieve better separation of closely migrating isoforms.

• Compare your results with known isoform expression patterns in different tissues or cell types.

• For definitive identification, consider combining antibody detection with mass spectrometry analysis or using isoform-specific antibodies if available.

• When studying isoform-specific functions, validate findings using complementary techniques such as RNA interference targeting specific isoforms or expression of tagged recombinant isoforms.

What are the best practices for using GATAD2A antibodies in chromatin immunoprecipitation (ChIP) experiments?

For optimal ChIP results with GATAD2A antibodies, follow these research-validated protocols:

How can I investigate GATAD2A interactions with other NuRD complex components?

The interaction between GATAD2A and other NuRD components is critical for understanding its functional role. Research has demonstrated that GATAD2A variants can disrupt interactions with CHD3, CHD4, and CHD5 . To study these interactions:

• Co-immunoprecipitation (Co-IP): Use GATAD2A antibodies for immunoprecipitation followed by Western blotting for other NuRD components. Both antibody products (#19388 and #17705) have been validated for IP applications .

• In vitro binding assays: Express FLAG-tagged CHD-CTD proteins together with HA-tagged GATAD2A proteins and perform pull-down assays with anti-FLAG resin, as demonstrated in research on GATAD2A variants .

• Proximity ligation assays: Visualize protein-protein interactions in situ using antibodies against GATAD2A and other NuRD components.

• Domain mapping: When studying specific variants or mutations, focus on the CR1 and CR2 conserved regions of GATAD2A, which are critical for interactions with MBD2/3 and CHD proteins .

• Functional consequences: Assess the impact of disrupted interactions on chromatin remodeling activity, gene expression, and cellular phenotypes.

How can GATAD2A antibodies be used in neurodevelopmental disorder research?

GATAD2A variants have been identified in individuals with neurodevelopmental disorders characterized by global developmental delay, structural brain defects, and craniofacial dysmorphology . Researchers studying these NuRDopathies can utilize GATAD2A antibodies in several ways:

• Patient-derived samples: Compare GATAD2A expression, localization, and complex formation in cells derived from affected individuals versus controls.

• Variant modeling: In cellular or animal models expressing GATAD2A variants (such as the de novo nonsense variant c.673C>T, p.R225*), use antibodies to assess protein expression, stability, and localization .

• Developmental studies: Track GATAD2A expression during neural development, particularly in models relevant to the structural brain defects observed in affected individuals.

• Mechanistic investigations: Combine ChIP-seq and RNA-seq to identify genes misregulated due to GATAD2A dysfunction and correlate these with neurodevelopmental phenotypes.

• Therapeutic screening: Use GATAD2A antibodies to monitor the efficacy of potential therapeutic approaches aimed at correcting NuRD complex dysfunction.

What is known about the role of GATAD2A in viral infections, and how can antibodies help study this?

Recent research has revealed that circular RNA GATAD2A (circ-GATAD2A) promotes the replication of influenza A virus H1N1 in A549 cells by inhibiting autophagy . This finding opens up new research directions for GATAD2A in viral infection contexts:

• Expression analysis: Use GATAD2A antibodies to monitor protein expression changes during viral infection and compare with circ-GATAD2A levels.

• Mechanistic studies: Investigate whether linear GATAD2A protein and circ-GATAD2A have related or distinct functions in autophagy regulation during viral infection.

• Protein-RNA interactions: Explore potential interactions between GATAD2A protein and its circular RNA form using RNA immunoprecipitation with GATAD2A antibodies.

• Therapeutic implications: Screen for compounds that modulate GATAD2A expression or function as potential antiviral strategies for influenza infections.

• Broader viral context: Extend studies to other viral infections to determine if the role of GATAD2A in autophagy regulation is specific to influenza or represents a more general antiviral mechanism.

What are common issues when using GATAD2A antibodies in Western blotting and how can they be resolved?

Researchers may encounter several challenges when using GATAD2A antibodies for Western blotting:

• Multiple bands: GATAD2A appears as two bands at 70 and 73 kDa due to different isoforms . If additional bands appear, optimize:

  • Blocking conditions (try 5% BSA instead of milk)

  • Antibody dilution (start with 1:1000 as recommended)

  • Washing stringency

• Weak signal: GATAD2A is a nuclear protein that may require specialized extraction:

  • Use nuclear extraction buffers containing appropriate detergents

  • Increase protein loading (up to 30-50 μg total protein)

  • Extend primary antibody incubation to overnight at 4°C

  • Use high-sensitivity ECL substrates

• High background: For cleaner results:

  • Increase blocking time and washing steps

  • Reduce primary antibody concentration

  • Try alternative blocking agents

  • Use freshly prepared buffers

• Batch variation: When comparing multiple experiments:

  • Include consistent positive controls

  • Consider using recombinant antibodies like E7B5B (#17705) which offer superior lot-to-lot consistency

  • Normalize GATAD2A signals to appropriate loading controls

How can I validate GATAD2A antibody specificity for my particular application?

Rigorous validation of antibody specificity is crucial for reliable research results:

• Genetic approaches:

  • Use CRISPR/Cas9-mediated GATAD2A knockout cells as negative controls

  • Employ siRNA knockdown to demonstrate reduced signal intensity

  • Express tagged GATAD2A and confirm co-detection with tag-specific antibodies

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide to block specific binding

  • Compare with non-competed antibody to identify specific versus non-specific signals

• Cross-validation:

  • Compare results from multiple GATAD2A antibodies targeting different epitopes

  • Cross-reference with mass spectrometry data from immunoprecipitated samples

• Species-specific validation:

  • For cross-species applications, confirm reactivity in each species of interest

  • Compare observed molecular weights with species-specific predictions

• Application-specific controls:

  • For ChIP, include IgG controls and validate enrichment at known GATAD2A binding sites

  • For IP, confirm enrichment of known GATAD2A interacting partners

How can GATAD2A antibodies contribute to stem cell and reprogramming research?

GATAD2A plays a non-redundant role in early stem cell differentiation, and its ablation enhances pluripotent reprogramming . Researchers exploring these functions can utilize GATAD2A antibodies to:

• Track differentiation dynamics: Monitor GATAD2A expression changes during stem cell differentiation through immunofluorescence or Western blotting.

• Study chromatin reorganization: Use ChIP-seq to map GATAD2A binding sites during pluripotency maintenance versus differentiation, correlating with chromatin accessibility and histone modification changes.

• Assess NuRD complex remodeling: Investigate how NuRD complex composition and GATAD2A-specific interactions change during cellular reprogramming.

• Functional interrogation: In GATAD2A knockdown or overexpression systems, use antibodies to confirm alteration of protein levels and correlate with reprogramming efficiency.

• Patient-specific iPSCs: When studying GATAD2A variants in patient-derived induced pluripotent stem cells, antibodies can help characterize protein expression, localization, and function in various differentiation contexts.

What are the potential applications of GATAD2A antibodies in autoimmune research?

While GATAD2A has not been directly implicated in autoimmune conditions in the provided search results, recent studies have identified widespread autoantibody production in multiple sclerosis (MS) and other autoimmune disorders . GATAD2A antibodies could contribute to this emerging research area by:

• Screening for autoantibodies: Determine if GATAD2A is targeted by autoantibodies in various autoimmune conditions by using purified GATAD2A protein in immunoassays.

• Epigenetic regulation in immune cells: Investigate GATAD2A and NuRD complex function in T cells, B cells, and other immune populations relevant to autoimmunity.

• Response to immunomodulatory therapies: Assess whether treatments like interferon-β-1a (used in MS) affect GATAD2A expression or chromatin binding patterns in immune cells.

• Biomarker development: Explore whether GATAD2A expression patterns correlate with disease activity or treatment response in autoimmune conditions.

• Functional consequences: Determine if alterations in GATAD2A function affect immune cell differentiation or cytokine production relevant to autoimmune pathogenesis.

What key research papers should I review before designing experiments with GATAD2A antibodies?

The following foundational literature provides crucial insights for researchers working with GATAD2A:

• Brackertz et al. (2002) - Original characterization of GATAD2A (p66α) as a component of the MeCP1 complex

• Brackertz et al. (2006) - Functional analysis of GATAD2A domains and their interactions with MBD2/3

• Gnanapragasam et al. (2011) - Role of GATAD2A in gene regulation through the NuRD complex

• Desai et al. (2015) - Structural insights into GATAD2A function in chromatin remodeling

• Gong et al. (2006) - Comparative analysis of GATAD2A and GATAD2B in the MBD2/NuRD complex

• Ding et al. (2017) - Neural development functions of GATAD2A

• Mor et al. (2018) and Zviran et al. (2019) - GATAD2A functions in stem cell biology and reprogramming

• Chen et al. (2023) - De novo variants in GATAD2A in individuals with neurodevelopmental disorders

This literature provides the foundation for understanding GATAD2A structure, function, and pathological relevance, which is essential for designing meaningful experiments and interpreting results obtained with GATAD2A antibodies.

What are the best methods for preserving GATAD2A antigenicity in fixed samples?

To maintain optimal GATAD2A antigenicity in fixed samples for immunohistochemistry or immunofluorescence:

• Fixation protocol:

  • For tissues: 4% paraformaldehyde for 24-48 hours followed by paraffin embedding

  • For cells: 4% paraformaldehyde for 10-15 minutes or ice-cold methanol for 10 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Optimize temperature and duration (typically 95-100°C for 20-30 minutes)

• Blocking and permeabilization:

  • Use 0.1-0.3% Triton X-100 for proper nuclear permeabilization

  • Block with 5-10% normal serum from the species of the secondary antibody

• Antibody incubation:

  • Extend primary antibody incubation to overnight at 4°C

  • Consider using amplification systems for low-abundance targets

• Controls and validation:

  • Include GATAD2A-overexpressing and knockdown samples as positive and negative controls

  • Confirm nuclear localization pattern consistent with chromatin-associated proteins