TRA2A Antibody

What is TRA2A and what cellular functions does it serve?

TRA2A is a transformer 2 alpha homolog protein that functions as an RNA splicing factor. The protein contains an RNA recognition motif (RRM) that enables it to bind to specific RNA sequences, particularly those with purine-rich nucleotide motifs . TRA2A is primarily localized in the nucleus, consistent with its role in RNA processing and splicing regulation. The protein has a molecular weight of approximately 33 kDa (282 amino acids) as calculated from its sequence, though it typically migrates at 33-35 kDa on SDS-PAGE gels . TRA2A participates in alternative splicing events that regulate gene expression patterns in both normal cellular functions and disease states. Recent studies have implicated TRA2A in cancer progression, particularly in esophageal cancer, where its upregulation correlates with poor prognosis .

What applications are TRA2A antibodies commonly used for?

TRA2A antibodies are utilized across multiple experimental applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemistry (IHC), and immunocytochemistry (ICC) . These antibodies are particularly valuable in coimmunoprecipitation (CoIP) experiments to identify protein-protein interactions, as demonstrated in studies examining TRA2A's association with viral ribonucleoproteins . RNA immunoprecipitation (RIP) assays using TRA2A antibodies have been instrumental in identifying RNA targets of TRA2A, revealing its binding to specific viral mRNAs during infection . Fluorescence colocalization studies using TRA2A antibodies have helped characterize its subcellular distribution and interaction with other nuclear proteins .

What species reactivity can be expected with commercial TRA2A antibodies?

Commercial TRA2A antibodies demonstrate variable species reactivity depending on the specific product. Based on the search results, typical reactivity profiles include:

The most broadly reactive antibodies target conserved epitopes, particularly in the C-terminal region of TRA2A . When selecting an antibody, researchers should verify reactivity with their specific model organism and validate cross-reactivity experimentally if working with unstated species .

How can TRA2A antibodies be used to study virus-host interactions?

TRA2A antibodies are invaluable tools for investigating the role of TRA2A in viral replication and host adaptation. Studies have shown that TRA2A interacts with the viral ribonucleoprotein (vRNP) complex during influenza virus infection . Specifically, researchers can employ TRA2A antibodies in the following experimental approaches:

• Coimmunoprecipitation (CoIP) assays: TRA2A antibodies have been used to pull down viral proteins such as PA, PB1, PB2, and NP in infected cells, revealing direct interactions between TRA2A and these viral components . For RNA-independent interactions, researchers should include RNase treatment controls to distinguish direct protein-protein interactions from RNA-mediated associations .

• Colocalization studies: Combining TRA2A antibodies with viral RNA detection (RNA FISH) or viral protein immunofluorescence enables the visualization of TRA2A's association with viral components in the nucleus . These studies have shown that TRA2A colocalizes with M vRNA and NP protein in the nucleus of infected cells .

• RNA immunoprecipitation (RIP): TRA2A antibodies can be used to isolate TRA2A-bound RNAs, revealing that TRA2A binds to specific viral mRNAs, such as YS-M mRNA in avian influenza and PR8-NS mRNA in human influenza viruses . This application is critical for understanding how TRA2A regulates viral RNA splicing and processing.

• Functional studies: In conjunction with TRA2A knockdown or overexpression, TRA2A antibodies can help monitor changes in viral protein expression patterns. Such studies have revealed that TRA2A has opposite effects on avian versus human influenza virus replication .

What role does TRA2A play in influenza virus host adaptation?

TRA2A has been identified as a crucial host factor that determines influenza A virus host adaptation through differential regulation of viral mRNA splicing . Research using TRA2A antibodies for detection and characterization has revealed:

• Opposite effects on different viral strains: Human TRA2A inhibits avian influenza virus replication (including H5N1, H9N2, and H7N9 strains) but promotes human influenza virus replication (H1N1 strains) . This differential effect makes TRA2A a key determinant in cross-species transmission of avian influenza.

• Mechanism of action: TRA2A binds to the intronic splicing silencer (ISS) motif in YS-M mRNA (from avian virus) and PR8-NS mRNA (from human virus), modulating splicing ratios in opposite directions . Specifically, TRA2A increases the M2/M1 ratio in avian virus infection while increasing the NEP/NS1 ratio in human virus infection .

• Species-specific activity: Importantly, chicken TRA2A (chTRA2A) does not exhibit the same effect on avian virus replication when expressed in human cells, highlighting the species-specific nature of this host adaptation mechanism .

To study these aspects, researchers should design experiments that combine TRA2A antibody detection with targeted knockdown or overexpression of TRA2A, followed by monitoring of viral replication kinetics and splicing patterns of viral mRNAs.

How can researchers explore TRA2A's role in cancer progression?

TRA2A has been implicated in cancer progression, particularly in esophageal cancer . Using TRA2A antibodies, researchers can design experiments to investigate:

How should researchers validate TRA2A antibody specificity?

Validation of TRA2A antibody specificity is crucial for experimental rigor and reproducibility. Comprehensive validation should include:

• Positive and negative controls:

  • Positive controls should include samples known to express TRA2A, such as A549 cells and mouse brain tissue, which have been experimentally verified to show positive signal .

  • Negative controls should include samples where TRA2A is either naturally absent or experimentally depleted through siRNA or shRNA knockdown .

• Molecular weight verification: A true positive signal should detect TRA2A at its expected molecular weight of 33-35 kDa in Western blot applications .

• Peptide competition assay: Pre-incubating the antibody with the immunogen peptide should abolish specific binding, confirming that the observed signal is due to recognition of the intended epitope .

• Orthogonal validation: Comparing results from antibodies targeting different epitopes of TRA2A can provide additional confirmation of specificity. For example, using antibodies that target the N-terminal region (AA 1-50) versus the C-terminal region in parallel experiments .

• Cross-validation with genetic approaches: Correlation between protein detection by antibody and mRNA levels measured by RT-PCR provides an additional layer of validation .

What are the optimal sample preparation methods for TRA2A detection?

Sample preparation significantly impacts the success of TRA2A detection across different applications. Based on published methodologies:

• For Western blotting:

  • Total protein extraction should use lysis buffers containing protease inhibitors to prevent degradation of TRA2A.

  • For nuclear-enriched fractions (where TRA2A predominantly localizes), nuclear extraction protocols yield better results than whole-cell lysates.

  • Denaturation at 95°C for 5 minutes in loading buffer containing SDS and DTT or β-mercaptoethanol ensures proper protein unfolding.

• For immunofluorescence:

  • Fixation with 4% paraformaldehyde preserves protein structure while allowing antibody accessibility.

  • Permeabilization with 0.1-0.5% Triton X-100 enables antibody penetration to detect nuclear TRA2A.

  • Blocking with 3-5% BSA or normal serum reduces non-specific binding.

• For coimmunoprecipitation:

  • Gentler lysis conditions using non-ionic detergents (like NP-40 or Triton X-100) at 0.5-1% concentrations help preserve protein-protein interactions.

  • When investigating RNA-independent interactions, include RNase treatment controls as demonstrated in studies of TRA2A interaction with viral proteins .

• For RNA immunoprecipitation:

  • Crosslinking with formaldehyde (1%) before lysis helps preserve RNA-protein interactions.

  • RNase inhibitors must be included in all buffers to prevent degradation of target RNAs.

  • Stringent washing conditions should be optimized to reduce background while maintaining specific interactions.

What controls are essential when using TRA2A antibodies in influenza research?

When studying TRA2A's role in influenza virus research, several critical controls should be included:

• For interaction studies:

  • IgG control antibodies should be used in parallel with TRA2A antibodies for immunoprecipitation to identify non-specific binding .

  • RNase treatment controls are essential to distinguish RNA-dependent from RNA-independent interactions, as demonstrated with TRA2A's interaction with NP (RNA-dependent) versus PB2 (RNA-independent) .

  • Input controls (5-10% of lysate) should be analyzed alongside immunoprecipitated samples to verify the presence of target proteins before pull-down.

• For functional studies:

  • Multiple siRNA or shRNA constructs targeting different regions of TRA2A should be used to rule out off-target effects .

  • Rescue experiments, where knockdown phenotypes are complemented by expressing siRNA-resistant TRA2A constructs, provide strong evidence for specific effects.

  • Species-specific controls, such as comparing human versus chicken TRA2A effects, can reveal host adaptation mechanisms .

• For virus replication assays:

  • Cell viability assays should be performed in parallel to ensure that observed effects on viral replication are not due to cytotoxicity from TRA2A manipulation .

  • Time-course experiments (e.g., 24, 36, 48, and 60 hours post-infection) help distinguish early versus late effects of TRA2A on viral replication .

  • Multiple virus strains (human H1N1, avian H5N1, H7N9, H9N2) should be tested to establish broad relevance of findings .

How can researchers address weak or absent signals in Western blotting?

When faced with weak or absent signals when using TRA2A antibodies in Western blotting, researchers should consider:

• Sample preparation optimization:

  • Ensure complete lysis and denaturation of samples, particularly for nuclear proteins like TRA2A.

  • Increase protein concentration by loading more total protein (up to 50-100 μg) or using enrichment methods like nuclear fractionation.

  • Verify protein transfer efficiency using reversible staining methods (Ponceau S) before antibody incubation.

• Antibody conditions:

  • Try reducing antibody dilution (e.g., from 1:8000 to 1:1000) if signal is weak .

  • Extend primary antibody incubation time to overnight at 4°C to enhance binding.

  • Test different blocking agents (BSA vs. non-fat milk) as some primary antibodies perform better with specific blockers.

• Detection system enhancement:

  • Switch to more sensitive detection methods (e.g., from colorimetric to chemiluminescent or enhanced chemiluminescent).

  • Increase exposure time when using film or adjust settings on digital imaging systems.

  • Consider using signal amplification systems if TRA2A is expressed at low levels.

• Sample-specific considerations:

  • Verify TRA2A expression in your specific sample type, as expression can vary across tissues and cell lines.

  • A549 cells and mouse brain tissue are confirmed to express detectable levels of TRA2A and can serve as positive controls .

What strategies can improve colocalization studies with TRA2A antibodies?

Colocalization studies examining TRA2A and viral proteins or RNA have provided valuable insights into TRA2A function . To optimize these experiments:

• Fixation and permeabilization optimization:

  • Test different fixatives (paraformaldehyde, methanol, or combinations) as they can affect epitope accessibility.

  • Adjust permeabilization conditions to ensure antibody access to nuclear proteins while preserving cellular structure.

• Antibody compatibility:

  • When performing double-labeling, ensure primary antibodies are from different host species to avoid cross-reactivity of secondary antibodies.

  • If using multiple rabbit antibodies, consider direct labeling of one antibody or sequential immunostaining with thorough blocking between steps.

• Signal-to-noise optimization:

  • Increase blocking duration (2-3 hours) with 3-5% BSA or normal serum matched to the host of the secondary antibody.

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffers to reduce background.

  • Use Sudan Black B (0.1-0.3%) treatment to reduce autofluorescence, particularly in tissues.

• Imaging considerations:

  • Use confocal microscopy rather than widefield to better resolve nuclear colocalization.

  • Acquire sequential scans rather than simultaneous acquisition to minimize bleed-through between channels.

  • Include single-labeled controls to set appropriate acquisition parameters and thresholds for colocalization analysis.

• Quantitative analysis:

  • Employ quantitative colocalization analysis using Pearson's correlation coefficient or Manders' overlap coefficient.

  • Analyze multiple cells (>30) across independent experiments to ensure statistical robustness.

How can researchers troubleshoot RNA immunoprecipitation issues with TRA2A antibodies?

RNA immunoprecipitation (RIP) using TRA2A antibodies has revealed specific binding to viral mRNAs . To address common challenges in RIP experiments:

• RNA integrity preservation:

  • Work quickly and maintain samples at 4°C throughout the procedure.

  • Add RNase inhibitors to all buffers at sufficient concentrations (40-100 U/mL).

  • Pre-chill all equipment and minimize sample handling time.

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-1%) and crosslinking times (5-15 minutes) to find optimal conditions.

  • Include a non-crosslinked control to assess the impact of crosslinking on antibody performance.

• Background reduction:

  • Pre-clear lysates with protein A/G beads before adding the TRA2A antibody.

  • Include extensive washing steps (4-6 washes) with buffers of increasing stringency.

  • Always include an IgG control antibody to identify non-specific RNA binding .

• RNA recovery and analysis:

  • Use carrier RNA or glycogen during RNA precipitation to improve recovery of low-abundance RNAs.

  • Confirm RNA quality using bioanalyzer or gel electrophoresis before RT-qPCR.

  • Include multiple reference RNAs (both positive TRA2A targets and negative controls) when designing RT-qPCR panels .

• Validation approaches:

  • Confirm results with multiple primer sets targeting different regions of the same RNA.

  • Perform reciprocal experiments using tagged TRA2A constructs with antibodies against the tag.

  • Validate binding sites through mutation analysis of predicted TRA2A binding motifs in target RNAs.