TRDMT1 Antibody, HRP conjugated

What is TRDMT1 and why is it significant for research applications?

TRDMT1, formerly known as DNMT2, is the first RNA cytosine methyltransferase identified in humans. It specifically methylates cytosine-38 in the anticodon loop of several tRNAs, including tRNA Asp-GUC, tRNA Gly-GCC, and tRNA Val-AAC . Initially misclassified as a DNA methyltransferase, TRDMT1's name was changed in 2006 to better reflect its biological function as an RNA methyltransferase .

TRDMT1 has been implicated in numerous critical biological processes:

• Regulation of inflammation through the TLR4-NF-κB/MAPK-TNF-α pathway

• Protection against oxidative stress, salt stress, and cellular senescence

• Regulation of homologous recombination in transcribed regions of the genome

• Control of cell proliferation and migration

These diverse functions make TRDMT1 a significant target for research in inflammatory disorders, stress response, and cancer biology.

What are the key molecular characteristics of TRDMT1 that researchers should consider?

TRDMT1 has several important molecular characteristics that researchers should consider when designing experiments:

• Molecular weight: The observed molecular weight in Western blot applications is approximately 45 kDa, though calculated weights range from 39-44 kDa depending on the isoform

• Domain structure: Contains motifs similar to DNA cytosine methyltransferases but functions specifically on tRNA

• Post-translational modifications: Subject to poly-ubiquitination at K251 by the E3 ligase TRIM28

• Mutation hotspots: G155 is a hotspot for somatic cancer mutations, with G155V increasing ubiquitination and reducing protein levels

• Functional residues: Specifically methylates the C5 position of C38 near the anticodon in target tRNAs

Understanding these characteristics is essential for interpreting experimental results and designing targeted research approaches.

How do TRDMT1 antibodies compare to other RNA methyltransferase antibodies?

When comparing TRDMT1 antibodies with other RNA methyltransferase antibodies, researchers should consider:

It's critical to validate antibody specificity when working with TRDMT1, as some commercial antibodies may cross-react with other methyltransferases due to structural similarities in catalytic domains .

What are the optimal conditions for Western blot detection of TRDMT1 using HRP-conjugated antibodies?

Based on technical data and published protocols, the following conditions are recommended for optimal Western blot detection of TRDMT1:

Researchers should always perform initial titration experiments to determine optimal antibody concentration for their specific experimental conditions and cell types.

How can researchers optimize immunofluorescence protocols for TRDMT1 localization studies?

For optimal immunofluorescence detection of TRDMT1:

• Fixation and permeabilization:

  • Use 4% paraformaldehyde fixation (10-15 minutes)

  • Permeabilize with 0.1% Triton X-100 for cytoplasmic access

  • Consider methanol fixation for certain epitopes

• Antibody application:

  • Primary antibody dilution: Begin with 1:100-1:500

  • Incubation time: Overnight at 4°C for optimal signal-to-noise ratio

  • Secondary antibody: Fluorophore-conjugated anti-species antibody at 1:1000

• Controls and validations:

  • Include TRDMT1 knockdown cells as negative controls

  • Use co-staining with known interacting partners to confirm specificity

  • Compare localization patterns with published data on tRNA processing bodies

• Special considerations:

  • TRDMT1 localization may change under stress conditions

  • DNA damage induction may cause relocalization to damage sites

  • RNA extraction procedures should be performed if correlating with RNA methylation studies

What flow cytometry protocols are most effective for intracellular TRDMT1 detection?

For effective intracellular TRDMT1 detection by flow cytometry:

• Cell preparation protocol:

  • Harvest cells in exponential growth phase

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS

• Antibody application:

  • Use 0.5 μg antibody per 10^6 cells

  • Incubate for 30-60 minutes at room temperature

  • For unconjugated primary antibodies, use appropriate fluorophore-conjugated secondary antibody

• Controls:

  • Include isotype control at equivalent concentration

  • Use TRDMT1 knockdown cells as biological negative control

  • Consider dual staining with cell cycle markers to assess cell cycle-dependent expression

• Analysis considerations:

  • Gate on single, viable cells

  • Compare mean fluorescence intensity across experimental conditions

  • Consider using median rather than mean for skewed distributions

How can TRDMT1 antibodies be used to study inflammatory pathways?

TRDMT1 plays a protective role in inflammation by regulating the TLR4-NF-κB/MAPK-TNF-α pathway . Researchers can utilize TRDMT1 antibodies to investigate this role through:

• Expression analysis in inflammation models:

  • Western blot analysis of TRDMT1 levels in LPS-treated tissues and cells

  • Immunohistochemistry of inflamed tissues to localize TRDMT1 expression

  • Correlation of TRDMT1 levels with inflammatory markers like TNF-α

• Mechanistic studies:

  • Co-immunoprecipitation to detect TRDMT1 interactions with TLR4 pathway components

  • Chromatin immunoprecipitation to identify inflammation-related genes regulated by TRDMT1

  • Phosphorylation analysis of p65 and p38 in relation to TRDMT1 expression levels

• Experimental design considerations:

  • Include time-course analysis as TRDMT1 expression significantly decreases in various tissues after LPS treatment

  • Compare wild-type and TRDMT1 knockout models to establish causality

  • Evaluate tissue-specific responses, as TRDMT1 shows variable expression patterns across tissues

Research has demonstrated that TRDMT1 knockout rats exhibit increased mortality, more severe tissue damage, and elevated TNF-α levels following LPS treatment , highlighting the importance of this protein in inflammatory regulation.

What is the role of TRDMT1 in DNA damage response, and how can antibodies help elucidate this function?

TRDMT1 has emerged as a key regulator of homologous recombination (HR) in transcribed genomic regions . Researchers can use antibodies to investigate this function through:

• Localization studies:

  • Track TRDMT1 recruitment to DNA damage sites using immunofluorescence

  • Co-localization with γH2AX and other DNA damage markers

  • Time-course analysis of TRDMT1 recruitment and removal from damage sites

• Regulatory mechanism analysis:

  • Detection of TRDMT1 ubiquitination at K251 by immunoprecipitation followed by ubiquitin blotting

  • Analysis of TRIM28 (KAP-1) interaction with TRDMT1 during DNA damage response

  • Comparison of wild-type TRDMT1 versus G155V mutant localization and stability

• Functional outcome assessment:

  • Correlation of TRDMT1 levels with sensitivity to DNA-damaging agents like cisplatin

  • Analysis of HR efficiency using reporter assays in TRDMT1-depleted cells

  • Evaluation of γH2AX clearance kinetics as a measure of repair efficiency

Research has shown that TRDMT1 depletion increases sensitivity to cisplatin in multiple cell lines including U2OS, MCF-7, and SKOV3 , suggesting a critical role in DNA damage repair and potential therapeutic applications in cancer treatment.

How can TRDMT1 antibodies be utilized in cancer research, particularly regarding therapy resistance?

TRDMT1 has significant implications in cancer research, especially regarding therapy resistance:

• Expression analysis in clinical samples:

  • Immunohistochemistry of patient tumor samples to correlate TRDMT1 expression with treatment outcomes

  • Tissue microarray analysis to compare TRDMT1 levels across cancer types and stages

  • Paired analysis of pre- and post-treatment samples to assess expression changes

• Mechanism of resistance studies:

  • Western blot analysis of TRDMT1 in sensitive versus resistant cell lines

  • Correlation of TRDMT1 levels with DNA repair capacity

  • Assessment of TRDMT1 G155V mutation status in responders versus non-responders

• Therapeutic targeting approaches:

  • Validation of TRDMT1 inhibitor efficacy using antibody-based detection of target engagement

  • Combination therapy studies correlating TRDMT1 inhibition with enhanced platinum sensitivity

  • Monitoring of TRDMT1 degradation in response to targeted therapies

Research has demonstrated that high expression of TRDMT1 in ovarian cancer correlates with platinum resistance, while the G155V mutation leads to hyper-ubiquitination, reduced TRDMT1 levels, and increased sensitivity to platinum therapy . This suggests TRDMT1 as both a biomarker for treatment response prediction and a potential therapeutic target.

What are common issues in TRDMT1 antibody applications and their solutions?

Researchers may encounter several challenges when working with TRDMT1 antibodies:

For HRP-conjugated antibodies specifically:

• Store at -20°C in small aliquots to prevent enzyme denaturation

• Avoid repeated freeze-thaw cycles that reduce HRP activity

• Include HRP inhibitors (e.g., sodium azide) in blocking solutions but not in antibody diluents

How can researchers validate TRDMT1 antibody specificity?

Thorough validation of TRDMT1 antibody specificity is critical for reliable results:

• Genetic validation approaches:

  • Western blot comparison of wild-type versus TRDMT1 knockout/knockdown samples

  • Overexpression of tagged TRDMT1 to confirm co-localization with antibody signal

  • siRNA-mediated knockdown with correlation to signal reduction

• Biochemical validation methods:

  • Peptide competition assays to confirm epitope specificity

  • Use of multiple antibodies targeting different TRDMT1 epitopes

  • Mass spectrometry validation of immunoprecipitated protein

• Cross-reactivity assessment:

  • Testing in multiple cell lines with known TRDMT1 expression levels

  • Comparing reactivity with related methyltransferases (especially DNMT1)

  • Verification of expected molecular weight and subcellular localization

• Application-specific validations:

  • For flow cytometry: Confirm signal reduction with TRDMT1 knockdown

  • For immunoprecipitation: Verify pulled-down protein by mass spectrometry

  • For immunohistochemistry: Include appropriate tissue controls and compare with mRNA expression data

What factors affect TRDMT1 protein expression and detection sensitivity?

Several factors can influence TRDMT1 expression and detection:

• Biological factors:

  • Tissue-specific expression patterns (variable across liver, lung, kidney, thymus)

  • Stress conditions significantly alter TRDMT1 levels (oxidative stress, inflammation)

  • Cell cycle phase may influence expression levels

  • Post-translational modifications, particularly ubiquitination at K251

• Technical factors:

  • Sample preparation methods affect epitope preservation

  • Lysis buffer composition impacts protein extraction efficiency

  • Storage conditions can lead to protein degradation

  • Fixation methods for immunohistochemistry/immunofluorescence alter epitope accessibility

• Genetic factors:

  • G155V mutation leads to increased ubiquitination and reduced protein levels

  • Other mutations may affect antibody binding without altering protein levels

  • Expression polymorphisms may cause variable baseline expression

• Experimental design considerations:

  • Include time-course analysis as TRDMT1 responds dynamically to stimuli

  • Compare multiple tissue/cell types as expression is heterogeneous

  • Consider both mRNA and protein analysis as post-transcriptional regulation is significant

How can TRDMT1 antibodies be used to study RNA methylation and its functional consequences?

TRDMT1's primary function is RNA methylation, and antibodies can help connect this biochemical activity to biological outcomes:

• Integrated methodological approaches:

  • RNA immunoprecipitation (RIP) using TRDMT1 antibodies to identify bound RNAs

  • Combined immunoprecipitation and RNA-BisSeq to correlate TRDMT1 binding with methylation status

  • Proximity ligation assays to detect TRDMT1 interactions with RNA processing machinery

• Functional analysis strategies:

  • Correlation of TRDMT1 protein levels with m5C in target RNAs

  • Analysis of translational efficiency changes in TRDMT1-depleted cells

  • Assessment of tRNA stability and fragmentation patterns

• Disease-relevant applications:

  • Comparison of TRDMT1 levels and RNA methylation in normal versus disease states

  • Investigation of stress-induced changes in TRDMT1 activity and RNA modification

  • Analysis of cancer-specific alterations in TRDMT1-mediated RNA methylation

Research has shown that TRDMT1 knockdown changes mRNA methylation levels and affects genes associated with cell cycle, RNA transport, and RNA degradation pathways . These findings suggest that TRDMT1-mediated RNA methylation has broader functional consequences beyond tRNA modification.

What is the potential of TRDMT1 as a therapeutic target, and how can antibodies advance this research?

TRDMT1 shows promise as a therapeutic target, particularly in cancer:

• Target validation approaches:

  • Immunohistochemical analysis of TRDMT1 expression in patient cohorts correlating with treatment outcomes

  • Western blot assessment of TRDMT1 levels before and after experimental therapies

  • Evaluation of TRDMT1 inhibitor efficacy using antibody-based detection methods

• Mechanistic investigations:

  • Analysis of TRDMT1 interactions with DNA repair machinery using co-immunoprecipitation

  • Assessment of RNA methylation changes following TRDMT1 inhibition

  • Monitoring cellular localization changes during drug treatment

• Therapeutic development strategies:

  • Screening assays using TRDMT1 antibodies to identify compounds that modulate protein levels

  • Combination therapy studies monitoring TRDMT1 expression/activity

  • Biomarker development for patient stratification based on TRDMT1 status

Research has demonstrated that a potent TRDMT1 inhibitor can resensitize TRDMT1-high tumor cells to cisplatin , suggesting practical therapeutic applications. Additionally, the G155V mutation associated with platinum sensitivity in ovarian cancer provides a potential biomarker for patient selection.