TADA2A Antibody

What is TADA2A and why is it important in transcriptional regulation?

TADA2A (also known as ADA2A, TADA2L) is a transcriptional activator adaptor protein that functions as part of the PCAF histone acetylase complex. It plays a crucial role in enhancing RNA polymerase II-mediated gene transcription by interacting with DNA-binding transcriptional activator proteins and the general transcription machinery at the basal promoter . TADA2A helps acetylate and destabilize nucleosomes, relieving chromatin constraints at promoters and facilitating transcription activation . This function makes TADA2A essential for understanding gene expression regulation mechanisms and chromatin remodeling processes in both normal cellular function and disease states.

What are the primary applications for TADA2A antibodies in research?

TADA2A antibodies are primarily utilized in Western Blot (WB) applications to detect and quantify TADA2A protein expression in various cell types and tissues . Based on available antibody specifications, most commercial TADA2A antibodies are validated for WB, with recommended dilutions typically ranging from 1:500 to 1:2,000 . Some antibodies are also validated for additional applications including ELISA, immunohistochemistry (IHC), and immunocytochemistry (ICC) . These applications enable researchers to investigate TADA2A expression patterns, protein-protein interactions, and functional roles in transcriptional regulation across different experimental contexts.

How should I optimize Western Blot protocols for TADA2A detection?

For optimal Western Blot detection of TADA2A:

• Sample Preparation: Use appropriate lysis buffers containing protease inhibitors to extract total protein from tissues or cell lines, with recommended loading of 25μg protein per lane .

• Antibody Selection: Choose rabbit polyclonal antibodies with validated reactivity to your species of interest (human, mouse, and rat are commonly validated) .

• Dilution Optimization: Begin with manufacturer-recommended dilutions (typically 1:500-1:2,000 for primary antibody) and optimize if necessary.

• Blocking Conditions: Use 3% non-fat dry milk in TBST as a blocking buffer to reduce background .

• Detection Method: Enhanced chemiluminescence (ECL) systems provide sensitive detection, with recommended exposure times of approximately 10 seconds for clear band visualization .

• Expected Band Size: Look for TADA2A at approximately 36 kDa based on predicted protein size .

• Controls: Include positive control lysates from cell lines with known TADA2A expression (BT-474, mouse brain, mouse skeletal muscle, or rat brain samples have been verified) .

How can I validate TADA2A antibody specificity for my experimental system?

Comprehensive validation of TADA2A antibody specificity requires:

• Epitope Analysis: Compare the immunogen sequence used to generate your antibody with your target species' TADA2A sequence. For example, some antibodies are raised against a synthetic peptide within the N-terminal region (MDRLGPFSNDPSDKPPCRGCSSYLMEPYIKCAECGPPPFFLCLQCFTRGF) , while others use recombinant fusion proteins containing amino acids 1-443 of human TADA2A (NP_001159577.2) .

• Knockout/Knockdown Controls: Implement TADA2A gene knockout or siRNA knockdown experiments to confirm antibody specificity by demonstrating reduced or absent signal.

• Peptide Competition Assay: Pre-incubate antibody with excess immunizing peptide before application to demonstrate signal reduction when epitope binding sites are blocked.

• Cross-Reactivity Assessment: Test antibody against samples from multiple species to confirm specified cross-reactivity claims (human, mouse, rat) and evaluate potential cross-reactivity with related proteins, particularly ADA2B/TADA2B, which shares sequence homology.

• Multiple Antibody Comparison: Validate results using different antibodies targeting distinct TADA2A epitopes to confirm consistent detection patterns.

What are the key considerations for studying TADA2A in chromatin regulation experiments?

When investigating TADA2A's role in chromatin regulation:

• Chromatin Immunoprecipitation (ChIP) Adaptation: Though not specifically validated for ChIP in the provided data, TADA2A antibodies may be adapted for ChIP experiments to identify chromatin regions where TADA2A functions. This typically requires:

  • Extensive protocol optimization

  • Higher antibody concentrations (3-5× WB concentrations)

  • Rigorous validation with appropriate controls

• Co-Immunoprecipitation (Co-IP) Analysis: Design experiments to investigate TADA2A's interactions with PCAF complex components and other transcriptional machinery proteins.

• Functional Studies: Combine antibody-based detection with functional assays measuring histone acetylase activity to correlate TADA2A presence with enzymatic function.

• Cell-Type Specificity: Account for potential variations in TADA2A expression and function across different cell types when designing experiments and interpreting results.

• Context-Dependent Regulation: Consider that TADA2A's functions may vary depending on cellular context, developmental stage, or disease state.

How should I design experiments to investigate TADA2A's role in transcriptional complexes?

To effectively study TADA2A's role in transcriptional complexes:

• Proximity Ligation Assays (PLA): Use TADA2A antibodies in conjunction with antibodies against known interaction partners to visualize and quantify protein-protein interactions in situ.

• Immunofluorescence Co-localization: Combine TADA2A antibody labeling with markers for nuclear transcription factories or specific chromatin states to analyze spatial organization.

• Sequential ChIP (ChIP-reChIP): For advanced applications, perform sequential ChIP using TADA2A antibodies followed by antibodies against other PCAF complex components to identify genomic regions where complete complexes assemble.

• Functional Domain Analysis: Design experiments incorporating antibodies recognizing specific TADA2A domains to determine their roles in complex assembly and function.

• Dynamic Interaction Studies: Implement live-cell imaging techniques with fluorescently tagged antibody fragments to study the dynamics of TADA2A recruitment to transcriptional complexes in real-time.

How can I resolve inconsistent TADA2A antibody performance in Western blot applications?

When encountering inconsistent Western blot results with TADA2A antibodies:

• Sample Preparation Optimization:

  • Ensure complete protein denaturation using appropriate reducing agents

  • Optimize protein extraction method for nuclear proteins

  • Test different lysis buffers specifically designed for nuclear/chromatin-associated proteins

  • Include phosphatase inhibitors to preserve potential post-translational modifications

• Protocol Modifications:

  • Increase primary antibody incubation time (overnight at 4°C)

  • Test different blocking agents (BSA vs. milk) as milk proteins may interfere with some antibody-epitope interactions

  • Adjust transfer conditions for high molecular weight proteins if detecting potential TADA2A complexes

  • Consider using PVDF membranes instead of nitrocellulose for potentially better protein retention

• Storage and Handling:

  • Aliquot antibodies upon receipt to avoid freeze-thaw cycles

  • Ensure proper storage at -20°C as recommended

  • Monitor antibody stability (12-month shelf life is typical)

  • Add carrier proteins if diluting antibodies for long-term storage

• Band Verification Strategies:

  • Compare band patterns across multiple tissue/cell types

  • Verify molecular weight matches the predicted size (approximately 36 kDa)

  • Consider potential post-translational modifications that might alter apparent molecular weight

What approaches can resolve weak or nonspecific signals when detecting TADA2A?

To address weak or nonspecific signal issues:

• Signal Enhancement Techniques:

  • Implement signal amplification systems compatible with your detection method

  • Adjust exposure times during imaging (starting with recommended 10 seconds)

  • Try alternative secondary antibodies with higher sensitivity

  • Consider using biotin-streptavidin amplification systems

• Background Reduction Strategies:

  • Increase washing duration and frequency

  • Test alternative blocking reagents (commercial formulations designed to reduce background)

  • Implement gradient optimization of primary and secondary antibody concentrations

  • Pre-adsorb secondary antibodies with tissue/cell lysates from the experimental species

• Sample Quality Assessment:

  • Verify protein integrity using total protein stains

  • Implement positive controls with known TADA2A expression (BT-474, brain tissues)

  • Include housekeeping protein controls to confirm equal loading

  • Check for protein degradation patterns indicating sample quality issues

How can TADA2A antibodies be integrated into studies of epigenetic regulation mechanisms?

For epigenetic research applications:

• Chromatin State Analysis:

  • Combine TADA2A immunoprecipitation with mass spectrometry to identify associated histone modifications

  • Implement sequential ChIP to correlate TADA2A binding with specific histone marks

  • Design experiments to map TADA2A distribution relative to transcriptionally active/inactive chromatin regions

• Temporal Dynamics Investigation:

  • Use TADA2A antibodies in time-course experiments following transcriptional stimulation

  • Combine with pulse-chase approaches to study turnover rates of TADA2A-containing complexes

  • Implement synchronized cell systems to study cell cycle-dependent changes in TADA2A function

• Disease Model Applications:

  • Compare TADA2A localization and function between normal and disease-state tissues

  • Investigate how pathological conditions affect TADA2A complex assembly

  • Study TADA2A's role in disease-specific transcriptional dysregulation mechanisms

• Single-Cell Applications:

  • Adapt TADA2A antibodies for single-cell proteomics approaches

  • Implement high-resolution imaging to study cell-to-cell variability in TADA2A distribution

  • Correlate with single-cell transcriptomics to link TADA2A presence with gene expression patterns

What considerations are important when designing experiments using TADA2A antibodies in specialized cell types?

When studying TADA2A in specialized cell contexts:

• Neuronal Systems:

  • TADA2A antibodies have been validated in brain tissues , making them suitable for neuroscience research

  • Consider subcellular compartmentalization in neurons (nuclear vs. dendritic localization)

  • Design experiments to investigate potential roles in activity-dependent transcription

• Stem Cell Research:

  • Investigate TADA2A's potential roles in maintaining pluripotency gene expression programs

  • Study dynamic changes during differentiation processes

  • Consider interactions with stem cell-specific transcription factors

• Cancer Cell Models:

  • Compare TADA2A expression and localization between normal and malignant cells

  • Investigate potential alterations in TADA2A-containing complexes in cancer cells

  • Study roles in oncogene activation or tumor suppressor silencing mechanisms

• Primary Cell Considerations:

  • Optimize fixation conditions for primary cells which may differ from established cell lines

  • Account for potentially lower expression levels in primary cells

  • Consider tissue-specific isoform expression when selecting antibodies

How might TADA2A antibodies contribute to understanding protein-protein interaction networks in transcriptional regulation?

TADA2A antibodies can advance protein interaction research through:

• Interactome Analysis Methods:

  • Implement antibody-based proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to TADA2A

  • Use TADA2A antibodies for co-immunoprecipitation followed by mass spectrometry

  • Develop split-reporter systems incorporating TADA2A epitopes to study dynamic interactions

• Structural Biology Applications:

  • Use antibodies as tools for protein purification prior to structural analysis

  • Implement antibody labeling for single-particle cryo-EM studies of TADA2A-containing complexes

  • Design epitope-specific antibodies to probe structure-function relationships

• Interaction Dynamics:

  • Study how environmental stimuli affect TADA2A interaction networks

  • Investigate cell cycle-dependent changes in TADA2A complex composition

  • Examine how disease states alter TADA2A's interactions with transcriptional machinery

What are the key methodological considerations for adapting TADA2A antibodies to emerging single-cell technologies?

For integration with single-cell technologies:

• Single-Cell Proteomics Adaptation:

  • Optimize antibody concentrations for microfluidics-based single-cell protein analysis

  • Validate antibody performance in limited-input scenarios

  • Consider conjugation with mass cytometry tags for high-dimensional analysis

• Spatial Transcriptomics Integration:

  • Adapt TADA2A antibodies for combined protein-RNA localization studies

  • Validate antibody compatibility with tissue clearing protocols

  • Develop multiplexing strategies to correlate TADA2A localization with gene expression patterns

• Live-Cell Applications:

  • Explore antibody fragment generation (Fab, scFv) for live-cell applications

  • Optimize membrane permeabilization protocols for intranuclear antibody delivery

  • Consider aptamer alternatives for dynamic tracking of TADA2A in living cells