mettl26b Antibody

What is METTL26 and why is it significant for research?

METTL26 (methyltransferase like 26) is a human protein that may also be known by alternative designations including C16orf13, JFP2, and UPF0585 protein C16orf13. The protein has a molecular weight of approximately 22.6 kilodaltons . As a member of the methyltransferase-like family, METTL26 is hypothesized to play roles in RNA modification and potentially epigenetic regulation, making it an important target for research in cellular biology, molecular mechanisms, and disease pathology studies.

METTL26 has orthologs in several mammalian species including canine, porcine, monkey, mouse, and rat models, allowing for comparative studies across different organisms . The conservation of this protein across species suggests functional significance that warrants further investigation through antibody-based detection and analysis methods.

What research applications are METTL26 antibodies most commonly used for?

METTL26 antibodies are utilized in a variety of research applications, with the most common being:

• Western Blot (WB): For detecting and quantifying METTL26 protein in cellular lysates

• Immunocytochemistry (ICC): For studying cellular localization of METTL26

• Immunofluorescence (IF): For visualizing METTL26 distribution in fixed cells

• Flow Cytometry (FCM): For quantifying METTL26 expression in cell populations

• Immunohistochemistry (IHC): For detecting METTL26 in tissue sections

Each of these applications requires specific optimization protocols to ensure reliable and reproducible results. For Western blot applications, researchers typically use antibodies targeting the N-terminal region of METTL26, as this region appears to be particularly immunogenic and provides consistent detection results.

What species reactivity do commercially available METTL26 antibodies exhibit?

Commercial METTL26 antibodies demonstrate reactivity across multiple species, with varying degrees of cross-reactivity. Based on available product information, most METTL26 antibodies show reactivity with:

How should researchers validate the specificity of METTL26 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For METTL26 antibodies, a comprehensive validation approach should include:

• Knockout/Knockdown Controls:

  • Utilize CRISPR-Cas9 generated METTL26 knockout cell lines

  • Compare with siRNA or shRNA knockdown models

  • Observe disappearance of the signal at the expected molecular weight (~22.6 kDa)

• Overexpression Verification:

  • Transfect cells with METTL26 expression vectors

  • Confirm increased signal intensity corresponding to overexpression levels

  • Include tagged (e.g., FLAG, HA) constructs for dual verification

• Peptide Competition Assays:

  • Pre-incubate the antibody with purified METTL26 peptide

  • Confirm signal reduction in subsequent detection assays

  • Use gradient concentrations to establish specificity thresholds

• Cross-reactivity Assessment:

  • Test on related methyltransferase family members

  • Compare results across multiple species if working with non-human models

  • Document any non-specific binding for proper experimental interpretation

It's recommended to perform multiple validation techniques rather than relying on a single method, as each approach addresses different aspects of antibody specificity. Documentation of validation results should be maintained for reproducibility purposes and potential publication requirements.

What are the optimal conditions for using METTL26 antibodies in western blotting?

Western blotting with METTL26 antibodies requires careful optimization for reliable detection of this 22.6 kDa protein. Based on experimental experience, the following protocol elements are recommended:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states

  • Sonicate briefly to ensure complete lysis and DNA shearing

  • Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol

• Gel Electrophoresis:

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

  • Include molecular weight markers spanning 10-50 kDa range

  • Load 20-40 μg of total protein per lane for cell lysates

• Transfer Conditions:

  • Semi-dry transfer: 15V for 30-45 minutes

  • Wet transfer: 100V for 1 hour at 4°C

  • PVDF membranes often provide better results than nitrocellulose for METTL26 detection

• Blocking and Antibody Incubation:

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

  • Primary antibody dilutions typically range from 1:500 to 1:2000 depending on manufacturer

  • Incubate with primary antibody overnight at 4°C

  • Wash 3-5 times with TBST, 5 minutes each wash

  • Secondary antibody incubation for 1 hour at room temperature (1:5000-1:10000 dilution)

• Detection Considerations:

  • Enhanced chemiluminescence (ECL) detection systems provide good sensitivity

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging systems

  • Expected band at approximately 22.6 kDa

This protocol serves as a starting point and may require further optimization depending on the specific antibody, sample type, and experimental conditions.

What troubleshooting approaches should be used when METTL26 antibodies yield inconsistent results?

When encountering inconsistent results with METTL26 antibodies, systematic troubleshooting should address several key areas:

• Antibody-Specific Issues:

  • Test multiple antibody clones targeting different epitopes

  • Verify antibody storage conditions and avoid freeze-thaw cycles

  • Consider batch-to-batch variation and request validation data from manufacturers

  • Optimize antibody concentration through titration experiments

• Sample Preparation Concerns:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Monitor protein degradation with freshly prepared protease inhibitors

  • Quantify protein concentration accurately using reliable methods (BCA/Bradford)

  • Consider the impact of post-translational modifications on epitope recognition

• Technical Variables:

  • Standardize incubation times and temperatures across experiments

  • Verify buffer compositions and pH values

  • Ensure consistent transfer efficiency by using transfer controls

  • Monitor blocking efficiency to reduce background noise

• Data Analysis and Interpretation:

  • Use appropriate controls for normalization

  • Apply quantification methods consistently

  • Consider biological variability in METTL26 expression levels

  • Document all experimental conditions for systematic comparison

Systematic documentation of troubleshooting steps will help identify patterns and resolve inconsistencies more effectively.

How should researchers design experiments to study METTL26 localization and expression patterns?

Designing experiments to investigate METTL26 localization and expression requires careful planning and appropriate methodological approaches:

• Cellular Localization Studies:

  • Immunofluorescence with METTL26 antibodies combined with organelle markers

  • Subcellular fractionation followed by western blotting

  • Live-cell imaging with fluorescently tagged METTL26 constructs

  • Co-localization analysis using confocal microscopy and quantitative overlap metrics

• Expression Pattern Analysis:

  • qRT-PCR for mRNA expression levels across tissues/cell types

  • Western blotting for protein expression quantification

  • Immunohistochemistry for tissue distribution patterns

  • Flow cytometry for single-cell expression profiling in heterogeneous populations

• Temporal Expression Dynamics:

  • Time-course experiments following stimulation or differentiation

  • Cell-cycle synchronization to assess fluctuations during division

  • Developmental stage comparison in appropriate model systems

  • Inducible expression systems for controlled temporal studies

• Comparative Analysis Approach:

  • Multi-tissue/multi-cell line screening for expression mapping

  • Normal versus pathological tissue comparison

  • Species comparison for evolutionary conservation assessment

  • Correlation with other methyltransferase family members

For cellular localization studies using immunofluorescence, researchers should use appropriate controls including secondary antibody-only controls and peptide competition assays to confirm specificity of the observed localization patterns.

What considerations are important when selecting METTL26 antibodies for specific research applications?

Selecting the appropriate METTL26 antibody for specific research applications requires consideration of several key factors:

• Application-Specific Requirements:

  • Western Blot: Antibodies recognizing denatured epitopes, often targeting the N-terminal region

  • Immunoprecipitation: Antibodies with high affinity for native conformations

  • Immunohistochemistry: Antibodies validated for fixed tissue sections

  • ChIP applications: Antibodies capable of binding in crosslinked chromatin contexts

• Epitope Considerations:

  • N-terminal targeting antibodies are commonly available and effective for many applications

  • Consider epitope conservation if working with orthologous proteins in different species

  • Evaluate epitope accessibility in native versus denatured states

  • Review supplier information regarding the specific immunogen used

• Antibody Format Selection:

  • Monoclonal: Greater specificity and reproducibility between batches

  • Polyclonal: Potentially higher sensitivity by recognizing multiple epitopes

  • Conjugated: Direct fluorophore or enzyme-linked antibodies for simplified protocols

  • Fragment formats (e.g., Fab, scFv): For specialized applications with size constraints

• Validation Documentation:

  • Review validation data provided by manufacturers

  • Assess literature citations using the specific antibody clone

  • Consider antibodies validated across multiple applications if versatility is needed

  • Evaluate knockout/knockdown validation data when available

Before committing to large-scale experiments, researchers should conduct small-scale pilot studies to validate antibody performance in their specific experimental system and conditions.

How can researchers effectively quantify METTL26 expression levels in different experimental contexts?

Accurate quantification of METTL26 expression requires selecting appropriate methods based on experimental objectives and sample types:

• Protein-Level Quantification:

  • Western Blotting: Semi-quantitative analysis with densitometry

    • Use appropriate loading controls (GAPDH, β-actin, α-tubulin)

    • Establish linear detection range for accurate quantification

    • Employ fluorescent secondary antibodies for wider linear dynamic range

  • ELISA: Quantitative measurement of METTL26 in solution

    • Develop sandwich ELISA using two antibodies recognizing different epitopes

    • Include a standard curve with recombinant METTL26 protein

    • Optimize sample dilution to ensure measurements within the linear range

  • Mass Spectrometry: Absolute quantification using labeled peptide standards

    • Target unique METTL26 peptides for selective reaction monitoring

    • Use isotope-labeled internal standards for precise quantification

    • Apply data-independent acquisition methods for comprehensive profiling

• mRNA-Level Quantification:

  • RT-qPCR: Relative quantification of METTL26 transcripts

    • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

    • Validate primer efficiency using standard curves

    • Select appropriate reference genes stable under the experimental conditions

  • RNA-Seq: Genome-wide expression analysis including METTL26

    • Apply appropriate normalization methods (FPKM, TPM, etc.)

    • Consider splice variant analysis to identify potential isoforms

    • Validate key findings with targeted qPCR confirmation

• Cellular-Level Analysis:

  • Flow Cytometry: Single-cell quantification of METTL26 levels

    • Optimize cell fixation and permeabilization protocols

    • Include isotype controls to establish background fluorescence

    • Apply appropriate gating strategies for heterogeneous populations

  • Imaging Cytometry: Spatial and intensity analysis of METTL26 expression

    • Standardize image acquisition parameters across samples

    • Apply consistent thresholding in analysis software

    • Utilize automated cell segmentation for unbiased quantification

When comparing METTL26 expression across different experimental conditions, it's essential to maintain consistent protocols throughout sample preparation, detection, and analysis to ensure reliable comparative results.

What is currently known about METTL26's functional role in cellular pathways?

Current understanding of METTL26's functional role remains limited, but emerging research suggests several potential pathways and functions:

• Predicted Methyltransferase Activity:

  • As a member of the methyltransferase-like family, METTL26 is predicted to catalyze methylation reactions

  • Potential targets may include nucleic acids (RNA/DNA) or proteins

  • The specific methyl donor (likely S-adenosylmethionine) and acceptor substrates require further characterization

  • Structural analysis suggests catalytic domains similar to other methyltransferases

• Cellular Localization Patterns:

  • Preliminary immunofluorescence studies indicate both nuclear and cytoplasmic distribution

  • Nuclear localization suggests potential roles in gene regulation or nuclear RNA processing

  • Cytoplasmic presence may indicate involvement in cytoplasmic RNA modification or protein methylation

• Associated Protein Interactions:

  • Limited proteomics data suggests potential interactions with RNA-binding proteins

  • Possible associations with other epigenetic modifiers in multi-protein complexes

  • Interactions may be dynamically regulated by cellular conditions or developmental stages

• Expression Patterns:

  • Differential expression across tissues suggests tissue-specific functions

  • Developmental stage-specific expression profiles have been observed in some model systems

  • Expression changes in response to certain cellular stresses have been reported

Further research using techniques such as CRISPR-Cas9 knockout models, mass spectrometry-based interaction studies, and functional genomics approaches will be essential to fully elucidate METTL26's biological roles.

How can researchers integrate METTL26 antibody-based techniques with other methodologies for comprehensive functional studies?

Integrating multiple methodological approaches with antibody-based techniques provides more robust insights into METTL26 function:

• Multi-omics Integration Strategy:

  • Combine antibody-based proteomics with transcriptomics to correlate protein and mRNA levels

  • Integrate ChIP-seq (if METTL26 has DNA interactions) with RNA-seq to link genomic binding with expression outcomes

  • Correlate METTL26 localization data with metabolomic profiles to identify functional pathways

  • Use network analysis to place METTL26 in broader cellular pathways based on multiple data types

• Temporal and Spatial Analysis Integration:

  • Combine time-course antibody-based assays with live-cell imaging of fluorescently tagged METTL26

  • Integrate subcellular fractionation data with super-resolution microscopy for detailed localization

  • Correlate tissue-specific expression patterns with cell type-specific functional assays

  • Use developmental timing studies with antibody detection to understand temporal regulation

• Functional Genomics Integration:

  • Combine CRISPR-Cas9 METTL26 knockout with antibody-based validation

  • Integrate results from overexpression studies with interactome mapping using co-immunoprecipitation

  • Correlate phenotypic changes in modified cell lines with molecular changes detected by antibodies

  • Use rescue experiments with mutant METTL26 variants to pinpoint functional domains

• Disease Model Integration:

  • Apply antibody-based detection in normal versus disease tissues/cells

  • Correlate METTL26 expression/localization changes with disease progression markers

  • Integrate patient sample analysis with cellular model findings

  • Use animal models with tissue-specific analysis of METTL26 expression and function

A comprehensive research approach should include appropriate controls, technical replicates, and biological replicates to ensure robust and reproducible findings across these integrated methodologies.

What are the emerging techniques for studying protein-protein interactions involving METTL26?

Several cutting-edge techniques are becoming available for investigating METTL26's protein interaction network:

• Proximity-Based Labeling Methods:

  • BioID: Fusion of METTL26 with a biotin ligase (BirA*) to biotinylate proximal proteins

  • APEX2: Peroxidase-based proximity labeling for temporal interaction studies

  • TurboID: Enhanced biotin ligase for more efficient proximity labeling

  • Comparative analysis across different cellular compartments and conditions

• Advanced Co-Immunoprecipitation Approaches:

  • Tandem Affinity Purification (TAP-tag): Dual purification steps for higher specificity

  • RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins): For identifying co-factors

  • Cross-linking Immunoprecipitation (CLIP): For capturing transient interactions

  • Quantitative proteomics using isobaric labeling to compare interaction partners across conditions

• Live-Cell Interaction Techniques:

  • FRET (Förster Resonance Energy Transfer): For direct protein-protein interactions

  • Split-fluorescent protein complementation: To visualize interactions in real-time

  • Optogenetics-based approaches: For controlled induction of interactions

  • Single-molecule tracking to study dynamics of interaction complexes

• Structural and Biophysical Methods:

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): For mapping interaction interfaces

  • Surface Plasmon Resonance (SPR): To determine binding kinetics and affinities

  • Native Mass Spectrometry: For intact complex analysis

  • Cryo-EM: For structural determination of METTL26-containing complexes

When designing interaction studies for METTL26, researchers should consider using complementary techniques to overcome limitations of individual methods and provide corroborating evidence for identified interactions.

How do METTL26 antibody applications differ across various model organisms?

The application of METTL26 antibodies across different model organisms requires consideration of species-specific factors and experimental adaptations:

• Mouse Models (Mus musculus):

  • High sequence homology allows many human-targeted antibodies to cross-react

  • Well-established protocols for tissue preparation and fixation

  • Genetic manipulation models (knockout, knockin) available for specificity controls

  • Recommended dilutions may need optimization compared to human samples

• Rat Models (Rattus norvegicus):

  • Good cross-reactivity with many human-targeted antibodies

  • May require longer primary antibody incubation times for optimal signal

  • Perfusion fixation techniques improve antibody penetration in tissue sections

  • Autofluorescence reduction protocols often necessary for IF applications

• Non-human Primate Models:

  • Highest sequence homology with human METTL26, excellent cross-reactivity

  • Similar protocol parameters as human samples

  • Limited availability of genetic manipulation controls

  • Valuable for translational research application validation

• Alternative Model Organisms:

  • Canine models: Variable cross-reactivity requiring thorough validation

  • Porcine models: Emerging models with moderate antibody cross-reactivity

  • Zebrafish: Limited antibody options, may require custom antibody development

  • Drosophila: Significant sequence divergence, specialized antibodies needed

When adapting METTL26 antibody protocols across species, researchers should:

• Perform epitope sequence alignment analysis to predict cross-reactivity

• Test multiple antibody clones targeting different epitopes

• Include appropriate positive and negative controls from each species

• Optimize antibody concentrations and incubation conditions specifically for each model organism

What is the best approach for developing and validating custom METTL26 antibodies for specialized research applications?

Developing custom METTL26 antibodies for specialized applications requires a systematic approach to ensure specificity and functionality:

• Strategic Epitope Selection:

  • Analyze METTL26 sequence for antigenic regions using prediction algorithms

  • Consider species conservation for cross-reactivity potential

  • Avoid regions with post-translational modifications unless specifically targeting them

  • Select unique sequences not present in related proteins (especially other METTL family members)

  • Target functional domains for potential blocking antibodies

• Immunization and Production Strategy:

  • Peptide-based approach: 15-20 amino acid synthetic peptides conjugated to carrier proteins

  • Recombinant protein fragments: Expression of soluble domains for immunization

  • DNA immunization: For conformational epitopes in native protein structure

  • Consider multiple host species (rabbit, goat, chicken) for diverse antibody repertoires

• Comprehensive Validation Protocol:

  • Western blot against recombinant protein and endogenous METTL26

  • Immunoprecipitation efficiency testing

  • Immunofluorescence patterns compared with commercial antibodies

  • Testing on METTL26 knockout and overexpression systems

  • Peptide competition assays to confirm specificity

  • Cross-reactivity testing against related proteins

• Application-Specific Optimization:

  • Fixation compatibility testing for histology/cytology applications

  • Buffer compatibility screening for diverse applications

  • Epitope accessibility assessment in different sample preparation methods

  • Determination of optimal working concentrations for each application

The timeline for custom antibody development typically spans 3-6 months, with comprehensive validation requiring additional time. Researchers should plan accordingly and consider developing multiple antibodies simultaneously to increase success probability.

How can researchers effectively analyze contradictory results obtained with different METTL26 antibodies?

When faced with contradictory results using different METTL26 antibodies, researchers should implement a systematic troubleshooting and reconciliation approach:

• Technical Validation Assessment:

  • Compare epitope recognition sites between antibodies

  • Evaluate detection methods and sensitivity differences

  • Assess batch variations and storage conditions

  • Review application-specific optimizations for each antibody

• Biological Explanation Exploration:

  • Consider potential isoforms or splice variants recognized differentially

  • Investigate post-translational modifications affecting epitope recognition

  • Examine cell type-specific or context-dependent protein conformations

  • Assess potential protein complex formations masking certain epitopes

• Systematic Reconciliation Strategy:

  • Implement orthogonal detection methods independent of antibodies

  • Use genetic approaches (siRNA, CRISPR) to validate specificity

  • Perform epitope mapping to precisely identify recognition sites

  • Conduct side-by-side comparisons with standardized protocols

• Resolution Documentation and Reporting:

  • Document all variables between contradictory experiments

  • Generate a decision tree for antibody selection based on application

  • Consider reporting discrepancies to antibody manufacturers

  • Include detailed methods descriptions in publications to aid reproducibility