GATAD2B Antibody, Biotin conjugated

What is GATAD2B and what cellular functions does it perform?

GATAD2B (GATA zinc finger domain-containing protein 2B) functions primarily as a transcriptional repressor in mammalian cells. It serves as a critical component of the histone deacetylase NuRD (Nucleosome Remodeling Deacetylase) complex that participates in chromatin remodeling and gene silencing . The protein enhances MBD2-mediated repression mechanisms and requires the presence of its paralog GATAD2A for efficient repression activity . Research indicates that GATAD2B targets MBD3 to specific loci within the nucleus, suggesting its role in targeted gene silencing . Additionally, emerging evidence points to GATAD2B's involvement in synapse development, expanding its functional significance beyond simple transcriptional control .

What applications are best suited for biotin-conjugated GATAD2B antibodies?

Biotin-conjugated GATAD2B antibodies are particularly valuable in multiple research applications that require high sensitivity and flexible detection systems. These antibodies have demonstrated efficacy in enzyme-linked immunosorbent assays (ELISA), providing reliable detection of GATAD2B in complex protein mixtures . The biotin conjugation enables versatile secondary detection strategies through the high-affinity streptavidin-biotin interaction system. This makes these antibodies especially suitable for chromatin immunoprecipitation (ChIP) experiments, protein interaction studies, and immunohistochemistry applications where signal amplification is beneficial. The biotin tag facilitates clean detection with minimal background in Western blotting applications, as demonstrated in studies using HeLa cell lysates .

How does GATAD2B interact with the NuRD complex components?

GATAD2B functions within the NuRD complex through specific protein-protein interactions that are essential for chromatin remodeling and transcriptional repression. Within this multiprotein complex, GATAD2B operates in conjunction with its paralog GATAD2A, and both proteins are required for optimal repression activity . Biochemical studies have demonstrated that GATAD2B enhances MBD2-mediated repression through direct interactions . The protein also targets MBD3 to discrete loci in the nucleus, suggesting a role in positioning the NuRD complex at specific genomic regions . Recent proximity-dependent biotin identification (BioID) experiments have revealed that GATAD2B is among the closest components in protein complexes involving transcriptional regulators like NOTCH1, indicating potential cross-talk between different regulatory pathways .

What are the recommended validation steps for GATAD2B antibodies?

When validating GATAD2B antibodies for research applications, multiple complementary approaches should be implemented to ensure specificity and reliability. Initial validation should include Western blot analysis using positive control samples with known GATAD2B expression, such as HeLa cell lysates at varying concentrations (15-50 μg) to establish detection sensitivity . Immunofluorescence (IF) confirmation of nuclear localization is essential, as GATAD2B should predominantly display nuclear staining consistent with its function in transcriptional regulation . For biotin-conjugated antibodies specifically, researchers should verify effective streptavidin binding without steric hindrance by comparing detection efficiency against unconjugated primary antibody controls. Additionally, knockdown/knockout validation using siRNA or CRISPR-Cas9 systems provides compelling evidence of antibody specificity through the demonstration of signal reduction or elimination.

How can biotin-conjugated GATAD2B antibodies be employed in proximity-dependent labeling experiments?

Biotin-conjugated GATAD2B antibodies offer significant advantages in proximity-dependent labeling studies like BioID or APEX2-based approaches. When implementing these methodologies, researchers should first optimize antibody concentration to ensure sufficient biotinylation without saturating the system or creating nonspecific background . For BioID experiments specifically, expression vectors containing BioID2-GATAD2B fusion constructs should be transfected into appropriate cell lines, followed by validation using both immunofluorescence and Western blot analyses to confirm proper nuclear localization and expression levels . Biotin supplementation (typically 50 μM) should be administered 24 hours prior to cell harvesting, and subsequent streptavidin affinity purification will capture biotinylated proteins in proximity to GATAD2B . Tandem mass spectrometry analysis of purified samples can then identify the proximal interactome, revealing both known and novel interaction partners within chromatin-associated complexes .

What strategies can address specific challenges in detecting GATAD2B isoforms?

Detection of specific GATAD2B isoforms requires careful consideration of epitope selection and validation. Drawing from approaches used with related proteins like GATAD2A (which has documented isoforms of 68 kDa and 65 kDa) , researchers should employ antibodies targeting epitopes common to all isoforms when general GATAD2B detection is desired. For isoform-specific detection, custom antibodies against unique regions (often within the amino acids 50-100 region) provide the necessary specificity . Western blotting should be performed using gradient gels (4-12% Bis-Tris) to achieve optimal separation of closely-sized isoforms. When conducting quantitative analyses, researchers should implement loading controls specifically chosen to match the subcellular fraction being analyzed – histone H3 for nuclear fractions or lamin B1 for nuclear envelope fractions rather than cytoplasmic markers like β-actin . For particularly challenging detection scenarios, immunoprecipitation followed by Western blotting provides enhanced sensitivity for low-abundance isoforms.

How do GATAD2A and GATAD2B functionally cooperate in transcriptional repression mechanisms?

GATAD2A and GATAD2B exhibit complex functional cooperation within the NuRD complex to achieve efficient transcriptional repression. Both proteins enhance MBD2-mediated repression, but research indicates they are not simply redundant . Experimental evidence suggests that optimal repression requires the presence of both factors, indicating synergistic rather than merely additive effects . Mechanistically, GATAD2B appears to have unique roles in targeting MBD3 to discrete nuclear loci, whereas GATAD2A may have distinct targeting functions . Proximity-ligation assays (PLA) combined with chromatin immunoprecipitation sequencing (ChIP-seq) can reveal co-localization patterns and shared genomic binding sites. Chemical crosslinking experiments, similar to those described for NOTCH1 interactions, using homogeneous bifunctional cell-permeable cross-linkers like DSP (with 12-Å spacer arms), followed by co-immunoprecipitation, can identify direct interactions between these paralogs and their binding partners in live cells .

What methodological considerations are important when using biotin-conjugated GATAD2B antibodies in proximity-dependent studies?

When implementing proximity-dependent studies with biotin-conjugated GATAD2B antibodies, several critical methodological considerations must be addressed. First, researchers must establish appropriate negative controls by using BioID2 protein alone (without GATAD2B fusion) to distinguish specific interactions from background biotinylation . Experimental design should include biological triplicates to ensure statistical robustness of identified interactions . Subcellular localization must be carefully validated using immunofluorescence to confirm that fusion proteins maintain the expected nuclear localization pattern consistent with GATAD2B's native distribution . Western blot analysis should verify comparable expression levels and biotinylation efficiency across experimental samples to enable accurate quantitative comparisons . For data analysis, both the number of recovered peptides and their intensity should be considered when ranking proximity interactions; this approach successfully identified MAML1 and GATAD2B as close components of the Notch activation complex in previous studies .

What are the optimal fixation and permeabilization conditions for immunohistochemistry with biotin-conjugated GATAD2B antibodies?

Optimal detection of GATAD2B in tissue sections requires careful consideration of fixation and permeabilization parameters. For formalin-fixed paraffin-embedded (FFPE) sections, antigen retrieval is critical due to GATAD2B's nuclear localization and potential epitope masking during formaldehyde crosslinking . Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-98°C for 20 minutes has proven effective for exposing GATAD2B epitopes while preserving tissue morphology. When using biotin-conjugated antibodies, blocking endogenous biotin is essential to prevent false-positive signals, particularly in biotin-rich tissues like liver, kidney, and brain. This can be achieved using avidin-biotin blocking kits prior to antibody incubation. Additionally, permeabilization with 0.3% Triton X-100 in PBS for 10 minutes ensures nuclear penetration, allowing access to chromatin-associated GATAD2B. For frozen sections, shorter fixation times (10 minutes) with 4% paraformaldehyde are recommended to minimize epitope masking while maintaining structural integrity.

How can cross-reactivity issues be addressed when using GATAD2B antibodies in multi-protein complex studies?

Addressing cross-reactivity challenges when studying GATAD2B within multi-protein complexes like NuRD requires systematic validation approaches. First, researchers should perform comprehensive specificity testing through Western blot analysis against purified recombinant GATAD2A and GATAD2B proteins to quantify potential cross-reactivity, particularly important given the sequence similarity between these paralogs . For immunoprecipitation studies, sequential immunoprecipitation can effectively distinguish between true complex components and cross-reactive artifacts – first pulling down with GATAD2B antibodies, then re-immunoprecipitating with antibodies against suspected interaction partners. When conducting proteomic analyses, inclusion of GATAD2B knockout/knockdown controls provides definitive exclusion lists for nonspecific binding proteins. Modern proximity-dependent approaches like BioID offer advantages by identifying proteins based on spatial proximity rather than direct binding affinity, potentially reducing cross-reactivity concerns . Additionally, competition assays using blocking peptides corresponding to the immunogen sequence can verify signal specificity in applications like immunohistochemistry or ChIP, where complex protein mixtures increase cross-reactivity risk.

What are the recommended approaches for studying the GATAD2B interactome in different cellular contexts?

Studying the GATAD2B interactome across different cellular contexts requires tailored methodological approaches that address context-specific challenges. For actively dividing cells, synchronization protocols should be implemented to control for cell cycle-dependent interactions, as chromatin remodeling complexes like NuRD demonstrate dynamic associations throughout the cell cycle. Proximity-dependent biotin identification (BioID) has proven particularly effective for mapping GATAD2B interactions in live cells, with validation through biological triplicates and appropriate controls . This approach successfully identified 133 candidate interaction proteins in one study context and 435 in another, highlighting the importance of cellular context . For terminally differentiated cells like neurons, where GATAD2B may play roles in synapse development, viral transduction of BioID2-GATAD2B constructs provides more efficient delivery than traditional transfection . When studying tissue-specific interactions, researchers should implement in vivo chemical crosslinking with cell-permeable agents like DSP (3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester)) before tissue homogenization to preserve transient interactions that might otherwise be lost during extraction procedures .

What quantitative methods are most appropriate for measuring GATAD2B expression levels?

Quantitative assessment of GATAD2B expression requires careful selection of methodologies appropriate to experimental contexts. For protein-level quantification, Western blotting using biotin-conjugated GATAD2B antibodies enables precise densitometric analysis when paired with appropriate loading controls like histone H3 for nuclear extracts . Multiple antibody dilutions (0.04-1.0 μg/mL) should be tested to establish a linear detection range, as demonstrated in HeLa lysate experiments . For transcript-level measurements, RT-qPCR remains valuable when designed with primers spanning exon-exon junctions to distinguish between potential splice variants. When analyzing tissue samples or heterogeneous cell populations, quantitative immunohistochemistry using biotin-conjugated antibodies with streptavidin-HRP detection provides spatial information alongside relative expression levels. For absolute quantification, targeted proteomics approaches using selected reaction monitoring (SRM) mass spectrometry with stable isotope-labeled reference peptides unique to GATAD2B offer the highest precision. This approach is particularly valuable when comparing expression across different experimental conditions or cell types where relative methods may introduce normalization artifacts.

How does GATAD2B interact with the NOTCH1 signaling pathway based on recent proximity studies?

Recent proximity-dependent biotin identification (BioID) studies have revealed unexpected connections between GATAD2B and the NOTCH1 signaling pathway. Analysis of the nuclear interactome for NOTCH1 oncoprotein using BioID methodology identified GATAD2B as one of the closest components of the Notch activation complex, alongside MAML1 . This spatial proximity was validated through multiple experimental approaches, including in vivo chemical crosslinking and endogenous co-immunoprecipitation in MOLT-4 and MB157 cell lines, both of which express high levels of NICD protein with activating NOTCH1 mutations . The interaction was successfully captured using DSP (3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester)), a homogeneous bifunctional cell-permeable cross-linker with a 12-Å spacer arm, suggesting the proteins exist within nanometer-scale proximity in living cells . This unexpected association between a core NuRD complex component and NOTCH1 signaling suggests potential cross-regulatory mechanisms between chromatin remodeling and Notch-mediated transcriptional activation, opening new avenues for investigating how these pathways may cooperate or antagonize each other in development and disease contexts.

What role might GATAD2B play in neurodevelopmental disorders based on current research?

Emerging research suggests GATAD2B may play critical roles in neurodevelopment, with implications for understanding certain neurodevelopmental disorders. Studies indicate that GATAD2B may be involved in synapse development, pointing to specialized functions in neuronal contexts beyond its general chromatin remodeling activities . This neuronal involvement aligns with clinical observations of neurodevelopmental phenotypes associated with GATAD2B mutations or expression abnormalities. To investigate these connections further, researchers can implement biotin-conjugated GATAD2B antibodies in several strategic approaches. Immunohistochemical analyses of developmental brain tissue samples can map temporal and regional expression patterns during critical neurodevelopmental windows. Proximity labeling in neuronal cultures can identify neuron-specific interaction partners that might differ from those in other cell types. ChIP-seq studies using biotin-conjugated GATAD2B antibodies can identify neuronal target genes under GATAD2B regulation. Additionally, co-immunoprecipitation experiments in neuronal contexts may reveal interactions with other neurodevelopmental disorder-associated proteins, potentially establishing mechanistic links between chromatin regulation and synapse formation or maintenance.

How can biotin-conjugated GATAD2B antibodies facilitate chromatin interaction mapping?

Biotin-conjugated GATAD2B antibodies offer significant advantages for mapping chromatin interactions and genomic binding sites with high resolution and sensitivity. For ChIP-seq applications, these antibodies enable efficient single-step pulldown of GATAD2B-associated chromatin fragments using streptavidin-coated magnetic beads, reducing background compared to conventional two-step antibody ChIP procedures. When implementing this approach, researchers should sonicate chromatin to 200-300bp fragments and use biotinylated antibody concentrations between 2-5μg per reaction for optimal results. For more complex analyses like ChIP-reChIP (sequential ChIP), biotin-conjugated GATAD2B antibodies in the first immunoprecipitation step allow stringent elution conditions without antibody contamination in subsequent steps. This approach has proven particularly valuable for distinguishing genomic sites where GATAD2B co-localizes with other NuRD complex components like GATAD2A . For three-dimensional chromatin interaction studies, combining biotin-conjugated GATAD2B ChIP with chromosome conformation capture techniques (ChIP-3C or ChIP-4C) can reveal how GATAD2B-bound regions physically interact with other genomic loci, providing insights into the spatial organization of GATAD2B-regulated genes.

What are the latest methodological advances in studying GATAD2B post-translational modifications?

Recent methodological advances have significantly enhanced our ability to study post-translational modifications (PTMs) of GATAD2B that may regulate its function in different cellular contexts. Mass spectrometry-based approaches have emerged as the gold standard for comprehensive PTM mapping, with several refinements particularly relevant to GATAD2B analysis. Researchers should implement sequential enrichment strategies, first using biotin-conjugated GATAD2B antibodies for immunoprecipitation, followed by modification-specific enrichment (e.g., titanium dioxide for phosphopeptides or anti-ubiquitin antibodies for ubiquitinated peptides). Parallel reaction monitoring (PRM) mass spectrometry offers targeted quantification of specific modified peptides with high sensitivity, enabling detection of low-abundance PTMs. For site-specific functional studies, researchers can generate modification-specific antibodies against known GATAD2B modification sites, though these require rigorous validation against modification-mimetic and modification-deficient mutants. Proximity ligation assays (PLA) using biotin-conjugated GATAD2B antibodies paired with modification-specific antibodies provide powerful tools for visualizing when and where specific modifications occur in intact cells, offering spatial and temporal resolution not achievable with biochemical approaches alone.