mettl26 Antibody

What is METTL26 and what is its function in human cells?

METTL26 (methyltransferase like 26) is a protein encoded by the METTL26 gene in humans. It may also be referred to by alternative names including C16orf13, JFP2, and UPF0585 protein C16orf13. Structurally, METTL26 is a relatively small protein with a reported molecular mass of approximately 22.6 kilodaltons . While the exact function of METTL26 remains under investigation, its classification as a methyltransferase-like protein suggests potential involvement in methylation processes, which are critical for various cellular functions including gene expression regulation and protein modification. Recent research indicates possible roles in epigenetic regulation, though the specific substrates and pathways remain areas of active investigation.

What types of METTL26 antibodies are available for research applications?

Research-grade METTL26 antibodies are available in various formats to accommodate different experimental approaches:

For specificity considerations, antibodies targeting the N-terminal region of METTL26 are commonly used, with products like ARP69013_P050 demonstrating reactivity across multiple species including human, mouse, and rat samples . When selecting an antibody, researchers should consider their specific application requirements, target species, and whether specific protein domains need to be targeted.

What detection methods are compatible with METTL26 antibodies?

METTL26 antibodies have been validated for multiple detection methods with varying sensitivities:

• Western Blot (WB): Most commercial antibodies support this application with recommended dilutions ranging from 0.04-0.4 μg/mL

• Immunohistochemistry (IHC): Typically using dilutions of 1:20-1:50

• Immunofluorescence (IF): For subcellular localization studies

• Flow Cytometry (FCM): For analysis of METTL26 in individual cells

• ELISA: For quantitative protein detection

When designing experiments, it's important to optimize antibody concentration for each specific application and sample type to ensure optimal signal-to-noise ratio and reproducibility of results.

How should I design validation experiments for METTL26 antibodies?

A robust validation strategy for METTL26 antibodies should include:

• Specificity testing: Compare signal in samples with known METTL26 expression versus negative controls

• Knockdown/knockout verification: Use siRNA or CRISPR to reduce METTL26 expression and confirm corresponding reduction in antibody signal

• Cross-reactivity assessment: Test across multiple species if working with non-human models

• Multiple technique validation: Confirm consistent results across different methods (e.g., WB, IF, IP)

For Western blot validation specifically, use positive control lysates from tissues known to express METTL26, run appropriate molecular weight markers to confirm the expected 22.6 kDa band, and include secondary-only controls to rule out non-specific binding . For immunohistochemistry validation, include appropriate isotype controls and test the recommended dilution range (typically 1:20-1:50) to determine optimal signal-to-background ratio .

What are the critical parameters for optimizing Western blots with METTL26 antibodies?

Western blot optimization for METTL26 detection requires attention to several key parameters:

• Sample preparation: Complete lysis and denaturation are essential; standard RIPA or NP-40 buffers with protease inhibitors are generally effective

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Gel percentage: 12-15% polyacrylamide gels are optimal for resolving the 22.6 kDa METTL26 protein

• Transfer conditions: Semi-dry transfer systems using PVDF membranes often provide better results than nitrocellulose for this small protein

• Blocking: 5% non-fat dry milk or BSA in TBST (1 hour at room temperature)

• Primary antibody incubation: Use recommended dilutions (0.04-0.4 μg/mL) in blocking buffer

• Washing: Four 5-minute washes with TBST after both primary and secondary antibody incubations

• Detection method: Enhanced chemiluminescence (ECL) systems provide good sensitivity, though fluorescent secondary antibodies may offer better quantification capabilities

For challenging samples or weak signals, extending primary antibody incubation to overnight at 4°C may improve detection while maintaining specificity.

What protocols are recommended for immunofluorescence studies using METTL26 antibodies?

For optimal immunofluorescence staining of METTL26:

• Cell preparation: Culture cells on poly-L-lysine coated coverslips to 60-80% confluence

• Fixation: 4% paraformaldehyde (15 minutes at room temperature) preserves most epitopes while maintaining cellular architecture

• Permeabilization: 0.1% Triton X-100 in PBS (10 minutes)

• Blocking: 5% normal serum (matching secondary antibody host) with 1% BSA (1 hour)

• Primary antibody: Dilute according to manufacturer recommendations; for most METTL26 antibodies, 1:100-1:500 in blocking buffer (overnight at 4°C)

• Secondary antibody: Use species-appropriate fluorophore-conjugated secondary at 1:500-1:1000 (1 hour at room temperature, protected from light)

• Nuclear counterstain: DAPI (1:1000, 5 minutes)

• Mounting: Use anti-fade mounting medium to preserve fluorescence

Include appropriate controls such as secondary-only samples and consider co-staining with organelle markers to better characterize METTL26 subcellular localization. Multiple METTL26 antibodies are validated for immunofluorescence applications, allowing for confirmation of findings with independent antibodies .

How can METTL26 antibodies be used to investigate protein-protein interactions?

For investigating METTL26 protein interactions, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use 1-2 μg of METTL26 antibody per 500 μg of protein lysate

  • Pre-clear lysates with appropriate control IgG

  • Include both negative controls (IgG) and input samples

  • Confirm precipitation of METTL26 before probing for interaction partners

• Proximity Ligation Assay (PLA):

  • Requires antibodies from different host species for METTL26 and potential interacting protein

  • Provides in situ visualization of protein interactions with subcellular resolution

  • Particularly valuable for detecting transient or weak interactions

• FRET/BRET analysis:

  • Requires fluorophore-conjugated antibodies or expression of tagged fusion proteins

  • Provides dynamic information about interactions in living cells

  • Useful for monitoring interaction changes in response to stimuli

When analyzing potential interaction partners, consider the methyltransferase-like domain of METTL26 as a starting point for hypothesis generation, as this functional domain likely mediates many of its biological interactions with substrates or regulatory proteins.

What approaches can be used to study METTL26 in the context of DNA methylation research?

To investigate potential roles of METTL26 in DNA methylation processes:

• Chromatin Immunoprecipitation (ChIP):

  • Use highly specific METTL26 antibodies validated for ChIP applications

  • Optimize crosslinking conditions (1% formaldehyde for 10 minutes is a standard starting point)

  • Follow with next-generation sequencing (ChIP-seq) to identify genomic binding sites

  • Compare binding patterns with DNA methylation profiles using methyl-C capture sequencing (MCC-seq)

• Methylation-specific assays:

  • Combine METTL26 knockdown/overexpression with methyl-C capture sequencing (MCC-seq)

  • MCC-seq provides higher resolution than array-based methods and targets functional genomic regions

  • Analyze changes in methylation patterns following METTL26 modulation

• Functional studies:

  • Examine METTL26 recruitment to specific genomic regions using ChIP-qPCR

  • Correlate METTL26 binding with methylation status and gene expression

  • Investigate interaction with known DNA methyltransferases using co-IP with METTL26 antibodies

For comprehensive methylation analysis, MCC-seq offers advantages over traditional 450k arrays, including better coverage of regulatory regions and nearly 8-fold increase in profiled sites (3.7 million vs. 450,000) .

How can METTL26 antibodies be applied in tissue-specific expression profiling?

For comprehensive tissue expression profiling of METTL26:

• Tissue microarray (TMA) analysis:

  • Use immunohistochemistry-validated METTL26 antibodies at recommended dilutions (1:20-1:50)

  • Include multiple tissue types on a single slide for standardized comparison

  • Employ digital image analysis for quantitative assessment of expression levels

  • Consider double staining with cell-type specific markers to identify expressing populations

• Multi-omics correlation approach:

  • Combine immunohistochemistry data with RNA-seq and proteomics

  • Compare protein detection with transcript levels to identify post-transcriptional regulation

  • The Human Protein Atlas project has established workflows for such correlative analyses

• Single-cell analysis:

  • Use flow cytometry with METTL26 antibodies for quantitative expression analysis in heterogeneous tissues

  • Combine with cell-type specific markers for comprehensive mapping of expression patterns

  • Consider imaging flow cytometry for simultaneous quantification and subcellular localization

This approach allows for correlating METTL26 expression with tissue-specific functions and potential disease associations across normal and pathological samples.

What are common issues with METTL26 Western blots and how can they be resolved?

For the specific case of METTL26, which has a predicted molecular weight of 22.6 kDa, bands at unexpected molecular weights should be carefully evaluated for potential splice variants or post-translational modifications. When troubleshooting, it's advisable to compare results using N-terminal versus C-terminal targeting antibodies to help identify potential processing or cleavage events .

How should discrepancies between different antibody-based detection methods be interpreted?

When facing discrepancies between detection methods (e.g., positive Western blot but negative immunohistochemistry):

• Consider epitope accessibility:

  • Different fixation/preparation methods may mask or expose different epitopes

  • Denatured proteins (WB) present different epitopes than native proteins (IF/IHC)

  • Try multiple antibodies targeting different regions of METTL26

• Evaluate expression levels:

  • Western blot may detect low abundance proteins not visible by IHC/IF

  • Consider signal amplification methods for less sensitive techniques

• Confirm specificity:

  • Use genetic models (knockdown/knockout) to validate all detection methods

  • Compare results with orthogonal techniques (mass spectrometry, RNA-seq)

• Analyze subcellular localization effects:

  • Compartmentalization may affect detection in certain assays

  • Nuclear proteins may require specialized extraction for WB but may be readily detected by IF

Methodological reconciliation often involves optimization of each technique independently and critical evaluation of the biological context. When possible, validate findings with multiple antibodies from different suppliers or targeting different epitopes .

What statistical approaches are recommended for quantifying METTL26 expression changes?

For rigorous quantification of METTL26 expression changes:

• Western blot densitometry:

  • Normalize to appropriate loading controls (β-actin, GAPDH, total protein)

  • Use technical replicates (minimum n=3) and biological replicates

  • Apply ANOVA with post-hoc tests for multiple group comparisons

  • Use non-parametric tests (Mann-Whitney) for non-normally distributed data

• Immunohistochemistry quantification:

  • Employ digital image analysis with validated algorithms

  • Score multiple fields per sample (>5 fields recommended)

  • Use H-score or Allred scoring systems for semi-quantitative analysis

  • Apply appropriate statistical tests based on data distribution

• Flow cytometry analysis:

  • Report median fluorescence intensity rather than mean (less affected by outliers)

  • Use fold-change relative to isotype controls

  • Apply Kolmogorov-Smirnov test for distribution differences

• Multi-assay integration:

  • Employ normalization strategies to compare across platforms

  • Consider meta-analysis approaches for combining multiple experimental results

  • Use correlation analyses to assess consistency across methods

For all quantitative analyses, ensure appropriate controls, biological replicates (n≥3), and transparency in reporting statistical methods and significance thresholds.

How might METTL26 antibodies contribute to epigenetic research beyond DNA methylation?

METTL26 antibodies could advance epigenetic research through:

• Histone modification studies:

  • ChIP-seq experiments to identify potential associations between METTL26 and specific histone marks

  • Sequential ChIP (re-ChIP) to determine co-occupancy with histone-modifying enzymes

  • Investigation of potential methyltransferase activity toward histone substrates

• RNA modification research:

  • RNA immunoprecipitation (RIP) using METTL26 antibodies to identify bound RNA species

  • Investigation of potential roles in m6A, m1A, or other RNA methylation processes

  • Analysis of METTL26 association with known RNA methyltransferase complexes

• Integrative multi-omics approaches:

  • Correlation of METTL26 binding sites with multiple epigenetic marks

  • Investigation of potential roles in reader-writer-eraser dynamics of epigenetic regulation

  • Comparison with other METTL family proteins with established epigenetic functions

These approaches could reveal novel functional roles for METTL26 in the broader epigenetic landscape, potentially identifying new regulatory mechanisms in development, cellular differentiation, or disease contexts.

What technological advances might enhance METTL26 antibody applications in research?

Emerging technologies poised to enhance METTL26 antibody applications include:

• Single-cell proteomics:

  • Application of METTL26 antibodies in mass cytometry (CyTOF)

  • Integration with single-cell transcriptomics for multi-modal analysis

  • Development of advanced antibody-based single-cell spatial proteomics

• Super-resolution microscopy:

  • Implementation of METTL26 antibodies in STORM/PALM imaging

  • Analysis of nanoscale protein distribution and interaction networks

  • Combined with proximity labeling for functional protein neighborhood mapping

• Antibody engineering:

  • Development of recombinant antibody fragments with enhanced tissue penetration

  • Creation of bispecific antibodies for simultaneous targeting of METTL26 and interaction partners

  • Generation of conditional binding antibodies for temporal control of detection

• In vivo applications:

  • Development of cell-permeable antibodies or intrabodies for live-cell imaging

  • Conjugation with PET/SPECT tracers for non-invasive imaging in model organisms

  • Integration with optogenetic systems for light-controlled binding/unbinding

These technological advances could significantly expand the utility of METTL26 antibodies beyond current applications, enabling new insights into dynamic cellular processes and in vivo functions.