METTL2 Antibody

What is METTL2 and what biological function does it serve?

METTL2 (Methyltransferase-like 2) is a methyltransferase responsible for catalyzing the formation of 3-methylcytosine (m3C) modifications in the anticodon loop of specific arginine tRNA isoacceptors in mammals. This enzyme plays a critical role in the post-transcriptional modification of tRNAs, which may influence translation efficiency and fidelity. Research has shown that METTL2 exists as two highly similar paralogs in humans: METTL2A and METTL2B, which share approximately 99% sequence homology . These enzymes form complexes with DALRD3 (DALR anticodon binding domain containing 3) protein to recognize and modify particular arginine tRNAs, highlighting a specialized targeting mechanism for RNA modification .

What applications are METTL2 antibodies typically used for?

METTL2 antibodies are primarily employed in several key experimental applications:

• Western Blot (WB): Detecting METTL2 protein expression in cell lysates and tissue samples, with recommended dilutions typically ranging from 1:2000 to 1:10000 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Visualizing the subcellular localization of METTL2 proteins in fixed cells, with recommended dilutions ranging from 1:125 to 1:500 .

• ELISA: Quantifying METTL2 levels in various sample types .

• Immunoprecipitation: Isolating METTL2 and its binding partners for interaction studies.

METTL2 antibodies have been validated in multiple human cell lines including A549, HeLa, MCF-7, SH-SY5Y, and PC-3 cells, providing researchers with flexibility in experimental design across different cellular contexts .

How can I distinguish between METTL2A and METTL2B in my experiments?

Distinguishing between METTL2A and METTL2B presents a significant challenge due to their extreme sequence similarity (99% homology) . Most commercially available antibodies, including the 84257-1-RR antibody, detect both isoforms. For researchers requiring isoform-specific detection:

• RNA-level analysis: Use isoform-specific PCR primers targeting the few nucleotide differences between METTL2A and METTL2B transcripts.

• CRISPR/Cas9 knockout controls: Generate single-isoform knockout cell lines to validate antibody specificity.

• Mass spectrometry: Employ targeted proteomic approaches to identify unique peptides that differentiate between the isoforms.

• Epitope-tagged constructs: Utilize differentially tagged METTL2A and METTL2B constructs for overexpression studies when absolute isoform specificity is required.

It's important to note that for many functional studies, distinguishing between these highly similar paralogs may not be necessary, as they appear to share substantial functional redundancy .

What is the optimal sample preparation protocol for METTL2 detection by Western blot?

For optimal METTL2 detection by Western blot, follow these methodological steps:

• Cell lysis: Use RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) supplemented with protease inhibitors.

• Protein quantification: Determine protein concentration via Bradford or BCA assay.

• Sample preparation: Mix 20-30 μg of protein with Laemmli buffer containing β-mercaptoethanol.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal separation.

• Transfer conditions: Transfer to PVDF membrane at 100V for 90 minutes or 30V overnight.

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute METTL2 antibody 1:2000-1:10000 in blocking solution and incubate overnight at 4°C .

• Washing: Wash membrane 3× with TBST, 5 minutes each.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Develop using ECL reagent and image.

The expected molecular weight for METTL2 is approximately 36-43 kDa, which aligns with the calculated molecular weight of 36 kDa plus potential post-translational modifications .

What controls should I include when using METTL2 antibodies?

To ensure experimental rigor when working with METTL2 antibodies, incorporate the following controls:

For immunofluorescence experiments, additional controls such as DAPI nuclear staining and cytoskeletal markers (e.g., α-tubulin) should be included to assess cell morphology and provide localization reference points.

How should I optimize immunofluorescence protocols for METTL2 detection?

For optimal immunofluorescence detection of METTL2, follow these methodological recommendations:

• Cell preparation:

  • Culture cells on glass coverslips or in chamber slides

  • Ensure subconfluent growth (60-80%) for clear visualization

• Fixation options:

  • 4% paraformaldehyde (15 minutes, room temperature) for structural preservation

  • Ice-cold methanol (10 minutes, -20°C) for enhanced epitope accessibility

• Permeabilization:

  • 0.1-0.3% Triton X-100 in PBS (10 minutes, room temperature)

• Blocking:

  • 5% normal serum (from secondary antibody host species) with 0.3% Triton X-100 in PBS (1 hour)

• Primary antibody:

  • Dilute METTL2 antibody 1:125-1:500 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

• Washing:

  • 3× with PBS, 5 minutes each

• Secondary antibody:

  • Fluorophore-conjugated anti-rabbit antibody (1:500)

  • Incubate 1-2 hours at room temperature in the dark

• Nuclear counterstain:

  • DAPI (1 μg/ml, 5 minutes)

• Mounting:

  • Anti-fade mounting medium

For MCF-7 cells specifically, which have validated positive staining for METTL2 , use a slightly lower dilution (1:125) initially, then optimize based on signal-to-noise ratio.

How can I investigate METTL2-DALRD3 interactions in my research system?

To study the functionally significant interaction between METTL2 and DALRD3, consider implementing these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-METTL2 antibody to pull down the protein complex

  • Detect DALRD3 by Western blot in the immunoprecipitated material

  • Include appropriate controls (IgG, input lysate)

• Proximity Ligation Assay (PLA):

  • Visualize protein-protein interactions in situ

  • Requires primary antibodies from different species against METTL2 and DALRD3

• Expression constructs:

  • Generate Twin-Strep-tagged METTL2A/B and FLAG-tagged DALRD3 constructs as described in the literature

  • Co-express in suitable cell lines (e.g., 293T cells)

  • Perform affinity purification using streptactin resin

• Truncation analysis:

  • Create truncated versions of both proteins to map interaction domains

  • Clone constructs into appropriate expression vectors (e.g., pcDNA3.1-3xFLAG)

• Functional readouts:

  • Assess m3C modification levels in arginine tRNAs upon disruption of the interaction

  • Employ techniques like mass spectrometry or specialized RNA modification detection methods

Research has demonstrated that METTL2A/B forms complexes with DALRD3 that are essential for recognizing specific arginine tRNAs destined for m3C modification. DALRD3-deficient cells show nearly complete loss of m3C modification in arginine tRNAs, highlighting the biological significance of this interaction .

What methods can I use to detect m3C modifications in tRNAs when studying METTL2 function?

For comprehensive analysis of m3C modifications in tRNAs when investigating METTL2 function, consider these methodological approaches:

• Antibody-based detection:

  • m3C immunoprecipitation and sequencing (m3C-IP-seq)

  • Utilize antibodies specifically recognizing 3-methylcytidine

  • Validate antibody specificity through dot blot analysis with competition assays

• Mass spectrometry:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Quantify m3C levels with high sensitivity and specificity

  • Compare relative abundance across different RNA species

• Genetic manipulation approaches:

  • Generate METTL2 or DALRD3 knockout/knockdown cell lines

  • Assess changes in m3C modification patterns

  • Perform rescue experiments with wild-type or mutant constructs

• In vitro methylation assays:

  • Reconstitute m3C formation using purified components

  • Include METTL2-DALRD3 complexes and substrate tRNAs

  • Analyze tRNA sequence elements required for modification

• RNA modification detection by sequencing:

  • Chemical treatment methods that cause signature mutations at modified sites

  • Next-generation sequencing to identify modification sites at single-nucleotide resolution

Recent research has demonstrated that m3C is present in various RNA types but is particularly enriched in tRNAs . The development of specialized techniques like m3C-IP-seq has enabled researchers to profile this modification transcriptome-wide, providing valuable insights into its distribution and potential functions.

How do I interpret changes in METTL2 expression levels across different experimental conditions?

When analyzing METTL2 expression changes across experimental conditions, consider these methodological guidelines for interpretation:

• Expression level quantification:

  • Normalize METTL2 signal to appropriate loading controls

  • Use densitometry software for quantitative Western blot analysis

  • Calculate fold changes relative to control conditions

  • Perform statistical analysis across biological replicates (minimum n=3)

• Isoform considerations:

  • Remember that standard METTL2 antibodies detect both METTL2A and METTL2B

  • Expression changes may reflect alterations in either or both isoforms

  • When critical, confirm with isoform-specific qPCR

• Functional correlations:

  • Associate METTL2 expression changes with m3C modification levels

  • Examine effects on specific arginine tRNA isoacceptors known to be METTL2 substrates

  • Assess potential impacts on translation efficiency or accuracy

• Biological context interpretation:

  • Consider tissue/cell-specific expression patterns of METTL2

  • Examine co-expression patterns with DALRD3, as they function together

  • Evaluate whether expression changes translate to functional consequences

• Technical validation:

  • Verify expression changes using multiple methodologies (e.g., WB, qRT-PCR)

  • Exclude technical artifacts by running appropriate controls

  • Consider dose-response or time-course experiments to establish causality

When documented in publications, present METTL2 expression data with quantification across multiple biological replicates, accompanied by appropriate statistical analysis and functional correlation studies to provide comprehensive context for the observed changes.

What are common issues with METTL2 antibody detection and how can I resolve them?

When encountering problems with METTL2 antibody detection, consider these common issues and their methodological solutions:

For METTL2 specifically, researchers should note that the expected molecular weight range of 36-43 kDa reflects potential post-translational modifications of the 36 kDa core protein . Additionally, since the antibody detects both METTL2A and METTL2B isoforms, slight variations in band patterns may reflect differential expression of these highly similar proteins.

How can I validate the specificity of my METTL2 antibody?

To rigorously validate METTL2 antibody specificity, implement these methodological approaches:

• Genetic manipulation controls:

  • siRNA/shRNA knockdown of METTL2

  • CRISPR/Cas9 knockout of METTL2

  • Compare signal between wild-type and depleted samples

• Overexpression validation:

  • Transfect cells with METTL2 expression constructs

  • Compare signal intensity between transfected and non-transfected cells

  • Include epitope-tagged METTL2 with detection by tag-specific antibodies as cross-validation

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Observe signal reduction/elimination in peptide-blocked samples

• Multiple antibody validation:

  • Test different antibodies targeting distinct METTL2 epitopes

  • Compare detection patterns for consistency

• Cross-species validation:

  • If applicable, test antibody reactivity in samples from different species

  • Align with known sequence conservation data

• Application-specific validation:

  • For WB: Observe expected molecular weight (36-43 kDa)

  • For IF/ICC: Compare with known subcellular localization patterns

  • For IP: Verify enrichment by mass spectrometry

When conducting these validation experiments, always include positive control samples from cell lines known to express METTL2, such as A549, HeLa, MCF-7, SH-SY5Y, or PC-3 cells, which have been previously validated for METTL2 expression .

How should I design experiments to study the functional relationship between METTL2 and DALRD3?

To investigate the functional relationship between METTL2 and DALRD3, design experiments following these methodological guidelines:

• Co-expression and interaction studies:

  • Generate expression constructs for both proteins (e.g., Strep-tagged METTL2A/B and FLAG-tagged DALRD3)

  • Perform co-immunoprecipitation to confirm physical interaction

  • Use truncation constructs to map interaction domains

  • Consider proximity ligation assays for in situ interaction visualization

• Functional dependency analysis:

  • Create single and double knockout/knockdown cell lines

  • Assess m3C modification levels in specific arginine tRNAs

  • Perform rescue experiments with wild-type or mutant constructs

  • Compare phenotypes between single and double depletions

• tRNA binding assays:

  • Analyze tRNA co-purification with METTL2 in the presence/absence of DALRD3

  • Examine specificity for different arginine tRNA isoacceptors

  • Determine sequence elements in tRNAs required for recognition

• Structure-function analysis:

  • Focus on the DALR anticodon binding domain in DALRD3

  • Investigate the methyltransferase domain in METTL2

  • Create point mutations in critical residues of each protein

• Reconstitution experiments:

  • Purify recombinant proteins

  • Perform in vitro methylation assays

  • Assess dependency of enzymatic activity on complex formation

Research has demonstrated that DALRD3 plays a crucial role in targeting METTL2 to specific arginine tRNAs. DALRD3-deficient cells show nearly complete loss of m3C modification in arginine tRNAs, and biochemical reconstitution experiments have confirmed that METTL2-DALRD3 complexes catalyze m3C formation in vitro . These findings highlight the functional interdependence of these proteins in the tRNA modification pathway.

How might METTL2-mediated tRNA modifications impact cellular translation and physiology?

The impacts of METTL2-mediated m3C tRNA modifications on cellular translation and physiology represent an emerging area of research with several potential implications:

• Translation fidelity:

  • m3C modifications in the anticodon loop may enhance codon-anticodon interactions

  • This could improve reading frame maintenance and reduce mistranslation events

  • Specific arginine codons may be decoded with different efficiencies depending on m3C status

• Translation efficiency:

  • Modified tRNAs may exhibit altered interactions with the ribosome

  • This could affect elongation rates for specific arginine codons

  • Global or selective effects on protein synthesis may result

• Stress response:

  • tRNA modification patterns might change under cellular stress conditions

  • METTL2 activity or expression could be regulated in response to environmental cues

  • m3C modifications might influence tRNA stability or fragmentation patterns

• Cell type-specific functions:

  • Differential expression of METTL2 and DALRD3 across tissues

  • Potential specialized roles in highly translating cell types

  • Disease-specific alterations in modification patterns

• Evolutionary conservation:

  • The METTL2-DALRD3 system represents a conserved mechanism for tRNA modification

  • Suggests fundamental importance in cellular biology

To investigate these aspects, researchers could employ ribosome profiling, RNA-Seq, proteomics, and targeted functional studies comparing wild-type and METTL2/DALRD3-deficient systems. Such approaches would help elucidate the biological significance of these tRNA modifications in various cellular contexts.

What are emerging techniques for studying METTL2 function beyond traditional antibody applications?

Beyond traditional antibody-based approaches, several cutting-edge methodologies are emerging for investigating METTL2 function:

• CRISPR-based approaches:

  • CRISPR interference (CRISPRi) for tunable repression

  • CRISPR activation (CRISPRa) for enhanced expression

  • Base editors for introducing specific mutations without double-strand breaks

  • Prime editing for precise genome modifications

• Advanced RNA modification detection:

  • m3C-IP-seq for transcriptome-wide profiling of m3C modifications

  • Nanopore direct RNA sequencing for native RNA modification detection

  • NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry) for dynamic modification analysis

• Structural biology approaches:

  • Cryo-EM analysis of METTL2-DALRD3-tRNA complexes

  • X-ray crystallography of component proteins and complexes

  • NMR studies of protein-RNA interactions

• Single-cell technologies:

  • Single-cell RNA-seq to examine cell-to-cell variation in METTL2 expression

  • Single-molecule imaging to visualize METTL2 localization and dynamics

  • Spatial transcriptomics to map METTL2 expression in tissue contexts

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and epitranscriptomics

  • Network analysis to position METTL2 within cellular pathways

  • Machine learning applications to predict functional impacts of m3C modifications

These emerging techniques offer powerful new ways to investigate the molecular mechanisms and biological significance of METTL2-mediated tRNA modifications, potentially revealing unexpected functions and regulatory connections in cellular physiology.

How do METTL2 expression patterns correlate with disease states and potential therapeutic applications?

The correlation between METTL2 expression patterns and disease states remains an active area of investigation with potential therapeutic implications:

• Cancer biology:

  • Altered translation programs are hallmarks of cancer progression

  • METTL2-mediated tRNA modifications may influence tumor cell protein synthesis

  • Expression patterns could vary across cancer types and stages

  • METTL2 has been detected in multiple cancer cell lines including A549 (lung), HeLa (cervical), MCF-7 (breast), and PC-3 (prostate)

• Neurodegenerative disorders:

  • Translation dysregulation is implicated in several neurodegenerative conditions

  • METTL2 is detected in neuronal cell lines such as SH-SY5Y

  • RNA modifications may influence neuron-specific protein synthesis

• Metabolic diseases:

  • Translation efficiency affects cellular energy utilization

  • tRNA modifications might coordinate metabolic adaptation

  • METTL2 activity could respond to cellular nutrient status

• Potential therapeutic strategies:

  • Small molecule modulators of METTL2 enzymatic activity

  • PROTAC (Proteolysis Targeting Chimera) approaches for targeted degradation

  • Antisense oligonucleotides for expression modulation

• Biomarker potential:

  • Expression levels as diagnostic or prognostic indicators

  • m3C modification patterns as functional readouts

  • METTL2-DALRD3 interaction status as disease markers

For investigating disease correlations, researchers should consider:

• Analyzing METTL2 expression across tissue and disease databases

• Examining genetic variants and their functional consequences

• Correlating expression with clinical outcomes in patient cohorts

• Developing models to test causality in disease-relevant systems

While direct therapeutic applications targeting METTL2 remain speculative, understanding its role in disease-specific contexts could reveal novel intervention points within the broader epitranscriptomics landscape.