TRMT2A Antibody

What is TRMT2A and what cellular functions does it perform?

TRMT2A is a dedicated mammalian enzyme responsible for m5U (5-Methyluridine) formation at tRNA position 54. This RNA modification is one of the most abundant modifications found in cytosolic tRNA . Recent research has revealed that TRMT2A's functionality extends beyond simple tRNA modification. Studies have demonstrated that TRMT2A contributes significantly to translation fidelity, with its knockdown resulting in reduced translational accuracy . Additionally, while 87% of cellular TRMT2A targets are tRNAs, it also interacts with other RNA types including HIST1H4B mRNA, KCND2 mRNA, and small subunit rRNA . It's worth noting that its paralog TRMT2B exhibits dual functionality in methylating both mitochondrial tRNAs and mitochondrial 12S rRNA .

What applications are TRMT2A antibodies most commonly used for?

TRMT2A antibodies are primarily utilized in Western Blotting (WB), Immunofluorescence (IF), and Flow Cytometry (FACS) applications . These applications allow researchers to detect and quantify TRMT2A protein expression in various experimental setups. Specifically, antibodies like the monoclonal mouse anti-TRMT2A (clone 1H2) have been validated at specific dilutions: Flow Cytometry (1:100), Immunofluorescence (1:100), and Western Blotting (1:4000) . When selecting a TRMT2A antibody, researchers should consider the specific application needs and validated dilution ratios to ensure optimal experimental outcomes.

How can TRMT2A antibodies be employed in studying translation fidelity mechanisms?

To investigate TRMT2A's role in translation fidelity, researchers can utilize TRMT2A antibodies in both loss-of-function and protein interaction studies. For loss-of-function experiments, begin with TRMT2A knockdown using siRNA or CRISPR-Cas9, then verify knockdown efficiency via Western blotting using anti-TRMT2A antibodies (1:4000 dilution recommended) . Next, assess translation fidelity through dual-luciferase reporter assays measuring frameshifting and stop codon readthrough rates. For interaction studies, employ co-immunoprecipitation with TRMT2A antibodies followed by mass spectrometry to identify binding partners involved in translation. Research has demonstrated that TRMT2A knockdown significantly reduces translation fidelity, suggesting its crucial role beyond simple tRNA modification . A complementary approach involves using TRMT2A antibodies in polysome profiling experiments to evaluate how TRMT2A depletion affects ribosome assembly and translation efficiency.

What experimental approaches can be used to investigate TRMT2A's interaction with different RNA types beyond tRNAs?

To comprehensively investigate TRMT2A's interaction with diverse RNA types, researchers should implement a multi-faceted approach combining immunoprecipitation and sequencing technologies. Begin with RNA immunoprecipitation (RIP) using TRMT2A antibodies to capture TRMT2A-bound RNAs. The immunoprecipitated material can be analyzed through next-generation sequencing (RIP-seq) to identify the variety of RNA targets. For higher resolution mapping of binding sites, utilize CLIP-seq (cross-linking immunoprecipitation followed by sequencing) or the more specialized FICC-CLIP (fluorouracil-induced-catalytic cross-linking–cross-linking and immunoprecipitation), which has previously revealed that 87% of TRMT2A targets are tRNAs, with the remaining targets including mRNAs (like HIST1H4B and KCND2) and rRNAs . To validate specific RNA-protein interactions, in vitro binding assays using electrophoretic mobility shift assay (EMSA) can be performed with recombinant TRMT2A and in vitro transcribed target RNAs, as demonstrated in previous studies where TRMT2A binding to various tRNAs was assessed with apparent KD values in the low nanomolar range .

How can researchers distinguish between TRMT2A and its paralog TRMT2B in experimental settings?

Distinguishing between TRMT2A and its paralog TRMT2B requires careful antibody selection and experimental design due to potential sequence homology. First, verify antibody specificity by examining the immunogen sequence used for antibody production—look for antibodies raised against regions with minimal sequence conservation between the paralogs. For instance, the described monoclonal antibody was raised against the full-length human recombinant TRMT2A protein . Conduct Western blot analysis with both positive controls (cells overexpressing each paralog individually) and negative controls (knockout cell lines) to confirm antibody specificity. For subcellular localization studies, perform immunofluorescence with TRMT2A antibodies (1:100 dilution) combined with mitochondrial markers, as TRMT2B primarily functions in mitochondria for m5U modification of mitochondrial tRNAs and 12S rRNA . Additionally, implement functional assays measuring methylation of cytosolic versus mitochondrial tRNAs to differentiate between the activities of these paralogs. When performing immunoprecipitation experiments, validate pulled-down proteins via mass spectrometry to ensure you've captured the intended target.

What are the optimal conditions for using TRMT2A antibodies in Western blotting applications?

For optimal Western blotting results with TRMT2A antibodies, careful attention to protocol details is essential. Begin with proper sample preparation: lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors, as TRMT2A may undergo post-translational modifications. Use fresh samples when possible, or store at -80°C with minimal freeze-thaw cycles. Load 20-40 μg of total protein per lane on a 10% SDS-PAGE gel, as TRMT2A is approximately 69 kDa. After electrophoresis, transfer to a PVDF membrane (recommended over nitrocellulose for this protein). Block with 5% non-fat milk in TBST for 1 hour at room temperature. For primary antibody incubation, use the monoclonal mouse anti-TRMT2A at 1:4000 dilution in blocking solution overnight at 4°C . After washing with TBST (3 × 10 minutes), apply HRP-conjugated anti-mouse secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature. Following thorough washing, develop using enhanced chemiluminescence. Include positive controls (e.g., HEK293T cells, which were used to produce the recombinant protein for antibody generation) and, if available, TRMT2A-knockout cells as negative controls to verify specificity.

What protocols should researchers follow when using TRMT2A antibodies for immunofluorescence studies?

For successful immunofluorescence studies with TRMT2A antibodies, follow this optimized protocol: Culture cells on glass coverslips coated with poly-L-lysine to enhance adherence. Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes. Block with 5% normal serum (from the same species as the secondary antibody) in PBS containing 0.1% Tween-20 for 1 hour. Incubate with mouse anti-TRMT2A primary antibody at 1:100 dilution in blocking buffer overnight at 4°C . After three 5-minute PBS washes, apply fluorophore-conjugated anti-mouse secondary antibody at 1:500 dilution for 1 hour at room temperature in the dark. For cellular localization studies, co-stain with markers for different cellular compartments, as TRMT2A has been shown to interact with proteins involved in RNA biogenesis . Particularly valuable is co-staining with nucleolar markers, as TRMT2A interacts with ribosomal components. Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes, wash, and mount slides with anti-fade mounting medium. Analyze using confocal microscopy to accurately assess subcellular localization. Include appropriate controls including primary antibody omission and competitive blocking with the immunizing peptide when available.

What considerations are important when using TRMT2A antibodies for flow cytometry?

When employing TRMT2A antibodies for flow cytometry, several technical considerations are crucial for generating reliable data. Begin with proper cell preparation: harvest 1×10^6 cells per sample, wash with cold PBS containing 2% FBS (FACS buffer), and fix with 2% paraformaldehyde for 15 minutes at room temperature if studying total TRMT2A levels. For intracellular staining, permeabilize cells with 0.1% saponin or commercial permeabilization buffer after fixation. Block with 5% normal serum in permeabilization buffer for 30 minutes. Incubate with mouse anti-TRMT2A antibody at 1:100 dilution for 45-60 minutes at 4°C . After washing twice with permeabilization buffer, apply fluorophore-conjugated secondary antibody at 1:500 dilution for 30 minutes at 4°C in the dark. Wash cells thoroughly and resuspend in FACS buffer for analysis. Include appropriate controls: isotype control (mouse IgG1) at matching concentration to assess background, and if possible, TRMT2A-knockdown samples as negative controls. For multiparameter analysis, consider combining TRMT2A staining with cell cycle markers to investigate connections between TRMT2A expression and cell proliferation, as TRMT2A has been described as a cell cycle regulator that suppresses proliferation .

What are the common issues when detecting TRMT2A in Western blotting and how can they be resolved?

Several challenges may arise when detecting TRMT2A through Western blotting. One common issue is weak or absent signal despite proper protocol execution. This may result from insufficient protein amount; increase loading to 40-50 μg of total protein or enrich samples through immunoprecipitation. Low TRMT2A expression in your cell type could also be responsible; verify expression levels in your model system through RT-qPCR before antibody-based detection. For non-specific bands, optimize antibody dilution (1:4000 is recommended based on validation data) , increase blocking stringency (5-10% milk/BSA), or try a different blocking agent. If high background is observed, increase washing duration and frequency (5 × 10 minutes with TBST) and ensure antibody dilutions are fresh. Multiple bands might indicate degradation products, post-translational modifications, or splice variants of TRMT2A; use freshly prepared samples with protease inhibitors and consider phosphatase inhibitors. For transfer issues, optimize transfer conditions for high molecular weight proteins (TRMT2A is ~69 kDa): use wet transfer at 30V overnight at 4°C. Finally, if the positive control works but your samples don't, consider that TRMT2A expression may be regulated under specific conditions in your experimental system.

How can researchers verify the specificity of their TRMT2A antibody results?

To rigorously verify TRMT2A antibody specificity, implement a multi-faceted validation approach. Begin with siRNA or CRISPR-Cas9 mediated knockdown/knockout of TRMT2A and confirm corresponding signal reduction in Western blotting, immunofluorescence, or flow cytometry. Overexpression of tagged TRMT2A (e.g., FLAG-tagged) allows for parallel detection with both anti-TRMT2A and anti-tag antibodies; co-localization confirms specificity. For further validation, perform peptide competition assays by pre-incubating the antibody with the immunizing peptide or recombinant TRMT2A protein prior to application; a true specific signal should be significantly reduced. Cross-reactivity testing is essential, particularly against TRMT2B due to potential sequence homology; run parallel experiments with cells expressing either paralog to ensure discrimination. Verify results across multiple cell lines with known varying levels of TRMT2A expression. For definitive validation, immunoprecipitate samples using the TRMT2A antibody and analyze by mass spectrometry to confirm the identity of the captured protein. Note that recent studies have employed such rigorous controls when mapping TRMT2A's interactome , providing a methodological template for comprehensive validation.

What challenges might researchers face when studying TRMT2A's interaction with tRNAs using immunoprecipitation methods?

Researchers investigating TRMT2A-tRNA interactions through immunoprecipitation face several technical challenges requiring methodological refinements. First, the transient nature of enzyme-substrate interactions makes capturing TRMT2A-tRNA complexes difficult; use crosslinking agents like formaldehyde (1-2%) or UV irradiation (254 nm) to stabilize these interactions prior to cell lysis. Consider using catalytically inactive TRMT2A mutants (such as mutations at the catalytic cysteine analogous to C324A in TrmA) to trap substrate complexes. RNA degradation during sample processing significantly impacts results; incorporate RNase inhibitors in all buffers and maintain samples at 4°C throughout the procedure. The relatively low binding specificity of TRMT2A for its targets (as demonstrated by similar KD values for various tRNAs in EMSA studies) may lead to enrichment of non-specific RNA interactions; implement stringent washing conditions and include competitor RNA like poly(U) during binding steps. For RIP-seq analysis, the co-immunoprecipitation of non-target RNAs associated with TRMT2A-interacting proteins rather than TRMT2A itself can confound results; perform parallel experiments with antibodies against known TRMT2A-interacting proteins to distinguish direct from indirect interactions. Finally, the dual functionality of TRMT2A in potentially methylating both tRNA and rRNA requires careful experimental design to distinguish between these target RNA classes.

How can TRMT2A antibodies be utilized to explore the enzyme's potential role in cancer biology?

TRMT2A antibodies offer multiple approaches to investigate its emerging role in cancer biology. Begin with immunohistochemistry on tissue microarrays from various cancer types, as TRMT2A overexpression has been correlated with HER+ positive breast cancer . Quantify expression levels through Western blotting across cancer cell line panels, comparing with normal tissue counterparts. For functional studies, combine TRMT2A knockdown/overexpression with antibody-based protein detection to monitor effects on proliferation, migration, and invasion in various cancer models. Since TRMT2A has been described as a cell cycle regulator that suppresses cell proliferation , use flow cytometry with TRMT2A antibodies to correlate its expression with cell cycle status in tumor samples. Investigate the molecular mechanisms by employing co-immunoprecipitation with TRMT2A antibodies to identify cancer-specific protein interactions. Explore TRMT2A's potential involvement in tRNA fragment (tRF) accumulation, as its knockout has been shown to affect tRF levels ; design experiments combining TRMT2A antibodies for protein quantification with small RNA sequencing for tRF profiling in cancer models. Determine if TRMT2A's role in translation fidelity contributes to cancer progression by assessing mistranslation rates in TRMT2A-manipulated cancer cells using reporter systems and validating TRMT2A levels via antibody-based methods.

What experimental approaches can investigate the relationship between TRMT2A and translation fidelity in disease models?

To investigate TRMT2A's role in translation fidelity within disease contexts, implement a comprehensive experimental strategy. First, establish disease-relevant cellular models (neurodegenerative disorders, cancer, etc.) and quantify TRMT2A expression levels via Western blotting using validated antibodies (1:4000 dilution) . For manipulation of TRMT2A levels, employ CRISPR-Cas9 for knockout, siRNA for knockdown, or expression vectors for overexpression, confirming changes with TRMT2A antibodies. Assess global translation fidelity using dual-luciferase reporters that measure frameshifting, stop codon readthrough, and misincorporation rates. For tissue-specific studies, perform immunohistochemistry with TRMT2A antibodies on disease-relevant tissues, correlating expression with pathological features. Employ ribosome profiling combined with TRMT2A immunoprecipitation to determine how TRMT2A affects ribosome positioning on mRNAs in disease states. Analyze the m5U modification status of tRNAs using mass spectrometry in relation to TRMT2A levels (verified by antibody detection). Since knockdown of TRMT2A has been shown to reduce translation fidelity , investigate whether disease-associated mutations in TRMT2A affect its enzymatic activity and protein-protein interactions using recombinant proteins and co-immunoprecipitation with TRMT2A antibodies. Finally, examine the therapeutic potential by testing whether restoring TRMT2A levels rescues translation fidelity defects in disease models.

How might advanced proteomics approaches utilizing TRMT2A antibodies reveal new insights about its interactome?

Advanced proteomics approaches leveraging TRMT2A antibodies can substantially expand our understanding of its cellular interactome and functions. Implement BioID or APEX proximity labeling by creating TRMT2A fusion proteins, followed by streptavidin pulldown and mass spectrometry to identify proteins in close proximity to TRMT2A in living cells. This approach overcomes limitations of traditional co-immunoprecipitation by capturing transient and weak interactions. For conventional immunoprecipitation, use crosslinking agents of varying lengths to stabilize different interaction types before pulldown with TRMT2A antibodies. Apply quantitative proteomics techniques like SILAC or TMT labeling to compare the TRMT2A interactome under different cellular conditions (e.g., stress, differentiation). Develop a TRMT2A-focused protein-protein interaction network by combining antibody-based pulldowns with mass spectrometry and validate key interactions through reciprocal co-immunoprecipitation and proximity ligation assays. Current research has already revealed that TRMT2A interacts with proteins involved in RNA biogenesis , but expanded proteomics studies could uncover connections to specific cellular pathways. Investigate dynamic changes in the TRMT2A interactome during cell cycle progression, given its reported role as a cell cycle regulator , using synchronized cells and time-course antibody-based pulldowns followed by mass spectrometry analysis. Finally, map post-translational modifications of TRMT2A through immunoprecipitation with specific antibodies followed by mass spectrometry to understand how these modifications might regulate TRMT2A's interactions and activities.