TARDBP Antibody

What is TARDBP and why is it important in research?

TARDBP, also known as TDP-43, is an RNA and DNA binding protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. It contains two RNA recognition motif (RRM) domains that are crucial for RNA processing and regulation. TARDBP is ubiquitously expressed throughout the body, with highest expression levels in the placenta, lung, pancreas, spleen, and genital tract. The protein plays vital roles in several cellular processes including binding to TAR DNA sequence motifs of HIV where it functions as a transcriptional repressor inhibiting HIV-1 transcription. Additionally, TARDBP is involved in splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, highlighting its importance in maintaining proper cellular function . Most notably, TARDBP has gained significant attention in research due to its association with neurodegenerative diseases as it is a major component of ubiquitin-positive inclusion bodies found in conditions like ALS and FTLD .

What types of TARDBP antibodies are available for research applications?

TARDBP antibodies are available in several formats to accommodate different experimental needs. These include mouse monoclonal antibodies and rabbit polyclonal antibodies that can detect TARDBP protein from various species including human, mouse, and rat samples. The antibodies are available in both non-conjugated forms and various conjugated formats including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates . Specific examples include the E-10 mouse monoclonal IgG2a kappa light chain antibody (sc-376311) which detects TARDBP across multiple species, and clone BSB-166, another mouse monoclonal antibody . The diversity of available antibodies allows researchers to select the most appropriate reagent based on their specific experimental design, target species, and detection method.

How do I determine the optimal dilution for my TARDBP antibody application?

Determining the optimal antibody dilution requires methodical testing and optimization for each specific application and sample type. For Western blotting applications, start with a range of dilutions (typically between 0.4-5 μg/mL as seen in validated protocols) using positive control samples like A431, HeLa, or RAW 264.7 cell lysates that are known to express TARDBP . For immunohistochemistry on paraffin-embedded tissue sections, a concentration range of 1.7-3 μg/mL has been validated for brain tissue samples . For immunofluorescence applications, start with 1-3 μg/mL as a working dilution . When optimizing, prepare a dilution series spanning at least three different concentrations (e.g., 1:500, 1:1000, 1:2000) and evaluate signal-to-noise ratio. The optimal dilution will provide strong specific staining of the target (generally nuclear localization for TARDBP in normal cells) with minimal background. Additional optimization may be necessary when changing experimental conditions, tissue types, or detection systems. Document all optimization steps and validated dilutions for reproducibility in future experiments.

What are the validated applications for TARDBP antibodies?

TARDBP antibodies have been validated for multiple experimental applications across different research contexts. Western blotting (WB) has been validated using human cell lines (A431, HeLa, K562, HepG2), mouse cell lines (RAW 264.7), and rat cell lines (NR8383) with specific bands detected at approximately 43-45 kDa under reducing conditions . Immunoprecipitation (IP) has been validated using HAP1 human near-haploid cell lines, demonstrating the ability to pull down TARDBP from complex protein mixtures . Immunofluorescence (IF) applications have been validated in fixed A431 human epithelial carcinoma cells and mouse splenocytes, with specific nuclear staining patterns observed . Immunohistochemistry with paraffin-embedded sections (IHCP) has been validated on human brain tissue (hippocampus and cortex) showing specific nuclear localization . Additionally, enzyme-linked immunosorbent assay (ELISA) applications have been validated for detecting human TARDBP in direct ELISA formats . For automated protein analysis, Simple Western™ systems have also been validated for detecting TARDBP in cell lysates . These diverse applications make TARDBP antibodies versatile tools for various research methodologies.

How should I prepare samples for optimal TARDBP detection by Western blot?

Optimal sample preparation for TARDBP Western blot analysis requires careful attention to protein preservation and extraction techniques. Begin by lysing cells or tissues in a buffer containing protease inhibitors to prevent protein degradation, which is particularly important for TARDBP due to its susceptibility to proteolytic cleavage. A recommended lysis buffer composition includes 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and a complete protease inhibitor cocktail. When working with tissues where TARDBP aggregates may be present (especially brain tissue from neurodegenerative disease models), consider sequential extraction methods using buffers of increasing solubilization strength to separate different protein fractions. For reducing conditions, add DTT (dithiothreitol) or β-mercaptoethanol to your sample buffer and heat samples at 95°C for 5 minutes before loading . Load 20-30 μg of total protein per lane for cell lines like A431, HeLa, or RAW 264.7 which express detectable levels of TARDBP. Transfer proteins to PVDF membranes (rather than nitrocellulose) for better protein retention and use Immunoblot Buffer Group 1 or 3 depending on your cell type as validated in published protocols . For detection, a specific band should be observed at approximately 43-45 kDa representing full-length TARDBP.

What controls should be included when using TARDBP antibodies in immunohistochemistry?

Robust control samples are essential for validating TARDBP antibody specificity in immunohistochemistry experiments. Include positive tissue controls known to express TARDBP such as human brain (cortex or hippocampus), breast, fallopian tube, testis, or skin samples . For disease-specific research, including tissue from patients with confirmed TDP-43 proteinopathies alongside age-matched controls can provide valuable comparison points. Negative controls should include tissues known to lack TARDBP expression or primary antibody omission tests on serial sections of your experimental tissue. Additionally, peptide competition assays, where the antibody is pre-incubated with purified TARDBP protein before application to tissue, can confirm binding specificity. For precise validation, include blocking peptide controls where available. When studying diseases with altered TARDBP localization (such as ALS or FTLD), include sections known to contain cytoplasmic TDP-43 aggregates alongside normal tissues with predominantly nuclear staining. This contrast will help validate the antibody's ability to detect pathological forms of the protein. Finally, consider dual-labeling with antibodies against different epitopes of TARDBP to further confirm specificity and potentially reveal differential detection of truncated or modified forms of the protein.

How can I distinguish between specific and non-specific bands in TARDBP Western blots?

Distinguishing between specific and non-specific bands in TARDBP Western blots requires systematic validation approaches. First, compare your observed band pattern with published literature; full-length TARDBP typically appears at approximately 43-45 kDa, though post-translational modifications or truncated forms may result in additional specific bands . Include positive control lysates from cell lines with confirmed TARDBP expression such as A431, HeLa, HepG2, K562 (human), RAW 264.7 (mouse), or NR8383 (rat) cell lines . Also consider running knockdown/knockout controls where TARDBP expression has been reduced or eliminated through siRNA or CRISPR/Cas9 methods—specific bands should show corresponding reduction in intensity. Peptide competition assays, where the antibody is pre-incubated with purified TARDBP protein before membrane incubation, can also help identify specific bands, which should diminish or disappear. For suspected cross-reactivity, use alternative antibodies targeting different TARDBP epitopes to confirm band patterns. When troubleshooting non-specific bands, optimize blocking conditions (consider switching between BSA and non-fat dry milk at 3-5%) and increase washing stringency. Adjusting antibody concentration and incubation time can also improve specificity. If working with brain tissue or disease models, be aware that C-terminal fragments (25-35 kDa) represent disease-specific cleavage products rather than non-specific bands .

What factors contribute to inconsistent TARDBP antibody performance across experiments?

Inconsistent TARDBP antibody performance can be attributed to several methodological and biological factors that require systematic troubleshooting. Antibody storage conditions significantly impact performance—repeated freeze-thaw cycles should be avoided by aliquoting the antibody upon first use, and storage temperature recommendations should be strictly followed. Different antibody lots may contain variations in activity and specificity, so maintaining detailed records of lot numbers and performing bridging experiments when transitioning between lots is advisable. Sample preparation variables also affect consistency; protein degradation during extraction, inconsistent fixation protocols, or variations in antigen retrieval methods can all alter epitope accessibility. For TARDBP specifically, its post-translational modifications (phosphorylation, ubiquitination, acetylation) may vary across experimental conditions and affect antibody recognition . When working with tissue samples, pre-analytical variables such as post-mortem interval and fixation duration critically impact immunoreactivity. Different detection systems (HRP-conjugated secondaries vs. fluorescent secondaries) have varying sensitivity thresholds and dynamic ranges. To enhance consistency, standardize all protocols including sample collection, processing times, buffer compositions, and incubation conditions. Incorporate internal controls in each experiment and consider using automated staining platforms for immunohistochemistry applications. Quantitative techniques like Western blotting should include loading controls and standard curves when possible.

How can TARDBP antibodies be used to study disease-specific protein modifications?

TARDBP antibodies can be strategically employed to investigate disease-specific modifications through complementary approaches targeting different protein states. For studying phosphorylation states, which are particularly relevant in ALS and FTLD where TARDBP is hyperphosphorylated, use phospho-specific antibodies alongside total TARDBP antibodies to calculate phosphorylation ratios . Immunoprecipitation with general TARDBP antibodies followed by immunoblotting with modification-specific antibodies (against phosphorylation, ubiquitination, acetylation, or SUMOylation sites) can reveal the proportion of modified protein in different experimental conditions. For detecting truncated forms of TARDBP common in pathological inclusions, combine antibodies targeting different epitopes (N-terminal vs. C-terminal) to identify specific fragmentation patterns. Sequential extraction protocols with buffers of increasing solubilization strength (e.g., low-salt, Triton X-100, sarkosyl, and urea fractions) followed by immunoblotting can differentiate soluble from aggregated TARDBP species. For microscopy applications, co-labeling with antibodies against TARDBP and specific modifications or aggregation markers (ubiquitin, p62, TIA-1) can reveal spatial relationships between protein states. Additionally, proximity ligation assays using TARDBP antibodies paired with modification-specific antibodies enable in situ visualization of modified protein with single-molecule sensitivity. When investigating novel modifications, mass spectrometry analysis of immunoprecipitated TARDBP can provide unbiased identification of post-translational modifications and their disease-associated changes.

What methodological approaches enable the study of TARDBP aggregation in disease models?

Studying TARDBP aggregation in disease models requires sophisticated methodological approaches spanning biochemical, microscopic, and functional analyses. Sequential extraction protocols represent a foundational biochemical approach, where tissues or cells are subjected to buffers with increasing solubilization strength (e.g., RIPA-soluble fraction followed by urea-soluble fraction) to separate normally soluble TARDBP from pathological aggregates. Western blotting of these fractions with TARDBP antibodies reveals distribution patterns between soluble and insoluble compartments . For microscopy-based approaches, immunohistochemistry using antibodies like clone BSB-166 can visualize changes in TARDBP subcellular localization, with nuclear clearing and cytoplasmic aggregation being hallmarks of pathology . Super-resolution microscopy techniques (STED, STORM) combined with appropriately conjugated TARDBP antibodies can reveal aggregate ultrastructure below the diffraction limit. Fluorescence recovery after photobleaching (FRAP) experiments using live-cell imaging with fluorescently tagged TARDBP can measure protein mobility changes associated with early aggregation. Filter trap assays, where cell lysates are passed through cellulose acetate membranes that trap large protein aggregates, followed by immunodetection with TARDBP antibodies, provide a quantitative measure of aggregation. For biochemical characterization, density gradient centrifugation of tissue homogenates followed by dot blotting with TARDBP antibodies can separate different sized aggregates. Additionally, conformation-specific antibodies that preferentially recognize misfolded TARDBP can be used alongside total TARDBP antibodies to distinguish pathological species from normal protein.

How can TARDBP antibodies be applied in multi-omics research approaches?

TARDBP antibodies can serve as powerful tools in multi-omics research by enabling the integration of protein-level data with transcriptomic, genomic, and metabolomic analyses. For proteomics applications, immunoprecipitation using validated TARDBP antibodies coupled with mass spectrometry (IP-MS) can identify the changing interactome of TARDBP under different conditions or disease states. Specifically, protein complexes can be isolated using antibodies like the mouse monoclonal E-10 (sc-376311) that has been validated for immunoprecipitation applications . In transcriptomic studies, ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) using TARDBP antibodies can map genome-wide DNA binding sites, while CLIP-seq (Cross-Linking Immunoprecipitation) can identify RNA binding targets of TARDBP, providing insight into its regulatory networks. For spatial multi-omics, multiplexed immunofluorescence combining TARDBP antibodies with markers of cellular stress, RNA metabolism, or neurodegeneration can correlate protein localization with cell state or pathology. Proximity-based biotinylation approaches (BioID, APEX) using TARDBP fusion proteins followed by streptavidin pulldown and proteomic analysis can map the spatial proteome surrounding TARDBP under different conditions. For metabolomics integration, correlative microscopy approaches combining TARDBP immunostaining with mass spectrometry imaging can reveal relationships between TARDBP aggregation and metabolic alterations in tissues. When analyzing single-cell multi-omics data, TARDBP antibodies can be used in CyTOF or CODEX platforms to correlate protein expression with transcriptomic clusters. For all these applications, antibody validation is crucial—confirming specificity using multiple antibodies targeting different epitopes enhances confidence in multi-omics findings.

How do TARDBP antibodies perform in detecting pathological inclusions in ALS and FTLD tissues?

TARDBP antibodies exhibit variable performance in detecting pathological inclusions in ALS and FTLD tissues, with success depending on epitope specificity, tissue preparation, and detection methods. In these neurodegenerative conditions, TARDBP undergoes post-translational modifications including hyperphosphorylation, ubiquitination, and C-terminal fragmentation before forming cytoplasmic inclusions . For optimal detection of these pathological structures, antibodies recognizing the C-terminal region of TARDBP are often more effective as this region is contained in the disease-associated fragments. Monoclonal antibodies like clone BSB-166 have been validated for detecting both nuclear and cytoplasmic localization in disease tissues . Tissue preparation significantly impacts detection sensitivity—short fixation times (24-48 hours) in 10% neutral buffered formalin followed by standard paraffin embedding typically yield optimal results. Antigen retrieval methods are crucial, with heat-induced epitope retrieval in citrate buffer (pH 6.0) often providing superior unmasking of TARDBP epitopes in inclusion bodies. For visualization, amplification systems such as polymer-based detection methods or tyramide signal amplification can enhance sensitivity for detecting smaller or less abundant inclusions. Fluorescent approaches allow co-localization studies with other pathological markers such as ubiquitin or p62. When quantifying pathology, consider both the number of inclusions and the degree of nuclear clearing, as the latter represents the depletion of normal nuclear TARDBP that contributes to disease pathogenesis through loss-of-function mechanisms.

What considerations are important when using TARDBP antibodies in cancer research?

When utilizing TARDBP antibodies in cancer research, several specific considerations must be addressed to generate reliable and interpretable data. TARDBP has been identified as having context-dependent roles in cancer biology, functioning as both an oncogene in some cancers (melanoma, glioblastoma) and a tumor suppressor in others (e.g., through miRNA miR-500a-3p in rhabdomyosarcomas) . Therefore, antibody selection should be guided by the specific cancer type under investigation. For studying TARDBP's role in cancer metabolism, antibodies detecting the full-length protein are generally appropriate as TARDBP regulates RNA associated with glucose and lipid metabolism . When investigating subcellular localization changes in cancer cells, immunofluorescence using antibodies validated for both nuclear and cytoplasmic detection is recommended, as TARDBP's function can vary based on its cellular compartmentalization. For quantitative assessments in cancer tissues, immunohistochemistry using antibodies like BSB-166 has been validated on diverse cancer types including lung adenocarcinoma . When studying TARDBP's interaction with miRNAs (such as miR-152, miR-500a-3p) in cancer cells, combining RNA-protein co-immunoprecipitation with TARDBP antibodies followed by qRT-PCR for specific miRNAs can reveal functional interactions. For high-throughput analyses, tissue microarray staining with carefully validated TARDBP antibodies can assess expression across multiple patient samples simultaneously. Cancer tissue heterogeneity necessitates analyzing multiple regions within tumors, and comparing TARDBP expression between tumor core, invasive front, and adjacent normal tissue can provide insights into its role in cancer progression.

How can TARDBP antibodies be used to investigate the protein's role in cellular stress responses?

TARDBP antibodies provide powerful tools for investigating the protein's dynamic behavior during cellular stress responses through multiple experimental approaches. Under stress conditions, TARDBP can relocalize from the nucleus to the cytoplasm and participate in stress granule formation—a process implicated in both adaptive stress responses and pathological aggregation in neurodegenerative diseases . To track this stress-induced translocation, live-cell imaging using fluorescently-tagged antibody fragments (Fabs) against TARDBP can monitor real-time localization changes in response to stressors such as oxidative stress, heat shock, or ER stress. For fixed-cell analyses, co-immunofluorescence using TARDBP antibodies alongside stress granule markers (G3BP1, TIA-1, eIF3) after stress induction reveals recruitment kinetics and composition of these membrane-less organelles. Biochemical fractionation of stressed cells into nuclear and cytoplasmic compartments followed by Western blotting with TARDBP antibodies provides quantitative assessment of translocation efficiency. To investigate posttranslational modifications associated with stress responses, immunoprecipitation with general TARDBP antibodies followed by modification-specific antibody detection (phospho-specific, ubiquitin-specific) can reveal stress-induced changes in protein state. For studying TARDBP's stress-related RNA targets, CLIP (Cross-Linking Immunoprecipitation) using validated TARDBP antibodies before and after stress exposure identifies dynamic changes in bound transcripts. When investigating the relationship between stress responses and disease pathology, proximity ligation assays combining TARDBP antibodies with antibodies against stress response proteins can visualize and quantify specific interactions that may be altered in disease states.