METTL25 Antibody

What is METTL25 and what is its known function?

METTL25 (Methyltransferase-like protein 25) is a human protein encoded by the METTL25 gene located on chromosome 12. It is also known by several synonyms including C12orf26 (Chromosome 12 open reading frame 26) and FLJ22789 . METTL25 functions as a putative methyltransferase, suggesting its involvement in methylation processes within cells . The full-length protein consists of 603 amino acids with a molecular weight of approximately 75.7 kDa . While the specific methylation targets and detailed molecular mechanisms remain under investigation, METTL25 belongs to the broader methyltransferase-like (METTL) family that plays crucial roles in epigenetic regulation and post-transcriptional modifications of various substrates.

Based on validated data, METTL25 antibodies have been successfully employed in several experimental applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of METTL25 in solution-based assays .

• Immunohistochemistry (IHC): For visualization of METTL25 expression in tissue sections, particularly useful in cancer research and pathology studies .

• Western Blotting (WB): For detection of METTL25 protein in cell or tissue lysates, allowing size determination and semi-quantitative analysis .

When designing experiments, researchers should consider the specific validation data available for each antibody and application. Proper controls should be included to ensure reliable interpretation of results .

What is the optimal storage condition for METTL25 antibodies?

For maximum stability and retention of activity, METTL25 antibodies should generally be stored at -20°C or -80°C . It is crucial to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody function . The rabbit polyclonal antibody is commonly supplied in a stabilizing buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . For working solutions, antibodies can be temporarily stored at 4°C for up to one week, but long-term storage should remain at recommended freezer temperatures. Always refer to manufacturer-specific guidelines for the particular antibody product being used.

How is METTL25 expression altered in hepatocellular carcinoma compared to normal tissue?

Research has shown that METTL family members, including METTL25, exhibit significant expression differences in hepatocellular carcinoma (HCC) compared to normal liver tissue. According to comprehensive analysis studies, most METTL proteins are upregulated in HCC tissues . Unlike the majority of METTL family members, METTL25 shows a unique expression pattern in relation to clinical features. While other METTLs (METTL1, 13, 18, 21A, 23, 2A, 2B, 5, 6, and 9) demonstrate associations with HCC stage and invasion depth (T), METTL25 did not show such correlations .

What are the best methods for validating METTL25 antibody specificity in experimental protocols?

Validating antibody specificity is crucial for obtaining reliable research results. For METTL25 antibodies, a multi-faceted validation approach is recommended:

• Knockout/Knockdown Controls: Compare antibody staining between wild-type samples and those with METTL25 gene knockout or knockdown. A specific antibody will show significantly reduced signal in the knockout/knockdown samples.

• Recombinant Protein Controls: Use purified recombinant METTL25 protein (such as the full-length human METTL25 protein described in search result 4) for competitive binding assays or as positive controls .

• Western Blot Analysis: Verify that the antibody detects a band of the expected molecular weight (approximately 75.7 kDa for full-length METTL25) . Multiple bands may indicate non-specific binding or protein degradation.

• Peptide Competition Assay: Pre-incubate the antibody with excess purified METTL25 protein or immunogen peptide before performing the primary application. Signal reduction confirms specificity to the target epitope.

• Cross-reactivity Testing: Test the antibody against related METTL family proteins to ensure it doesn't cross-react with structurally similar proteins, especially important when studying METTL25 in the context of other METTL family members .

• Multiple Antibody Validation: Compare results using different antibodies targeting distinct epitopes of METTL25. Concordant results strengthen confidence in specificity.

How does METTL25 compare to other METTL family members in terms of function and clinical significance in cancer?

The METTL family consists of several methyltransferase-like proteins with various functions in RNA modification, protein methylation, and epigenetic regulation. Based on the research data:

METTL25 stands out as having a distinct pattern compared to other family members, particularly in lacking association with HCC stage and invasion depth . Unlike METTL21A and METTL6, which have been identified as independent prognostic markers in HCC through multivariate studies, METTL25 does not demonstrate significant prognostic value . This suggests METTL25 may have different functional roles or regulatory mechanisms in carcinogenesis compared to other METTLs.

What experimental considerations should be made when using METTL25 antibodies for immunohistochemistry?

When performing immunohistochemistry (IHC) with METTL25 antibodies, several critical experimental factors should be considered:

• Tissue Fixation and Processing: Optimal fixation (typically 10% neutral buffered formalin for 24-48 hours) and proper processing are essential for preserving both tissue morphology and antigenicity. Overfixation can mask epitopes, while underfixation may compromise tissue integrity.

• Antigen Retrieval Method: Since METTL25 is an intracellular protein, effective antigen retrieval is crucial. Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be optimized based on the specific antibody recommendations.

• Antibody Selection: Both polyclonal and monoclonal antibodies against METTL25 are available for IHC applications . Polyclonal antibodies may provide enhanced sensitivity due to recognition of multiple epitopes, while monoclonal antibodies offer higher specificity.

• Antibody Concentration and Incubation Conditions: Titration experiments to determine optimal antibody dilution are essential. Typically, longer incubation times (overnight at 4°C) with lower antibody concentrations yield better signal-to-noise ratios than shorter incubations with higher concentrations.

• Detection System Selection: For low-abundance proteins like METTL25, amplification-based detection systems such as polymer-HRP or tyramide signal amplification may improve sensitivity compared to standard ABC methods.

• Controls: Include positive tissue controls (tissues known to express METTL25), negative tissue controls (tissues not expressing METTL25), and technical negative controls (primary antibody omission) in each IHC run.

• Multiplex Staining Considerations: When performing co-localization studies with other markers, ensure antibody compatibility regarding species origin and detection systems to avoid cross-reactivity.

• Quantification Methods: For research requiring quantitative assessment of METTL25 expression, establish consistent scoring criteria (e.g., H-score, percentage positive cells, or intensity scales) and employ multiple independent scorers to ensure reproducibility.

What is the role of METTL25 in the tumor microenvironment based on current research?

Research into the tumor microenvironment (TME) and METTL25's role within it is still emerging. Based on the available data, analysis of protein interaction networks including METTL25 and related genes has revealed potential involvement in TME remodeling . Single-cell sequencing approaches have been employed to construct protein interaction networks that include METTL25, suggesting its participation in cellular communication within the complex tumor ecosystem .

The METTL family proteins collectively influence various aspects of the TME, including:

• Immune Cell Infiltration: Analysis of associations between METTLs (including METTL25) and immune cell infiltration patterns suggests these proteins may modulate the immune component of the TME .

• Epithelial-Mesenchymal Transition (EMT): Research has examined connections between METTL expression and EMT processes, which are critical for cancer cell invasion and metastasis .

• Immune Checkpoint Regulation: Studies have investigated relationships between METTL proteins and immune checkpoint expression, with potential implications for immunotherapy responsiveness .

• Cancer Stem Cell Dynamics: METTL family proteins have been linked to cancer stem cell pathways, which contribute to tumor heterogeneity and treatment resistance .

• Drug Sensitivity Profiles: Correlations between METTL expression patterns and drug sensitivity have been observed, suggesting potential roles in treatment response prediction .

While specific mechanistic details of METTL25's individual contribution to these processes require further investigation, the current research framework provides direction for future studies exploring its functional significance in the TME and potential as a therapeutic target or biomarker.

How can researchers effectively optimize Western blot protocols for METTL25 detection?

Optimizing Western blot protocols for METTL25 detection requires attention to several key parameters:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for efficient protein extraction

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Determine optimal protein concentration (typically 20-50 μg total protein per lane)

  • Denature samples completely (95-100°C for 5 minutes in reducing sample buffer)

• Gel Selection and Separation:

  • Choose 8-10% SDS-PAGE gels for optimal resolution of METTL25 (75.7 kDa)

  • Consider gradient gels (4-15%) if analyzing multiple proteins of varying sizes

  • Run gel at 80-100V through stacking gel and 120-150V through resolving gel

• Transfer Optimization:

  • For proteins >70 kDa like METTL25, use wet transfer methods rather than semi-dry

  • Transfer at 30V overnight at 4°C for high molecular weight proteins

  • Include 10-20% methanol in transfer buffer for PVDF membranes; reduce to 10% for proteins >100 kDa

• Blocking Conditions:

  • Test both 5% non-fat dry milk and 3-5% BSA in TBST as blocking agents

  • Block for 1-2 hours at room temperature or overnight at 4°C

• Antibody Selection and Dilution:

  • For monoclonal antibodies like clone 2B6, start with 1:500 to 1:2000 dilution

  • For polyclonal antibodies, begin with 1:1000 dilution and optimize as needed

  • Incubate primary antibody overnight at 4°C for maximum sensitivity

• Detection System:

  • Enhanced chemiluminescence (ECL) detection systems provide good sensitivity

  • For low expression levels, consider using more sensitive ECL substrates (femto or pico level)

  • Fluorescent secondary antibodies offer quantitative advantages for densitometry

• Controls and Validation:

  • Include positive control (tissue/cell line known to express METTL25)

  • Include negative control (tissue/cell line with low/no METTL25 expression)

  • Use loading control appropriate for your experimental context (β-actin, GAPDH, etc.)

  • Consider using recombinant METTL25 protein as a reference standard

• Troubleshooting Multiple Bands:

  • If multiple bands appear, verify if they represent different isoforms, post-translational modifications, or degradation products

  • Peptide competition assay can help confirm specificity of detected bands

What are the recommended controls when performing ELISA with METTL25 antibodies?

When performing ELISA with METTL25 antibodies, implementing a robust set of controls is essential for reliable and interpretable results:

• Standard Curve Controls:

  • Use purified recombinant METTL25 protein to generate a standard curve spanning the expected detection range

  • Include at least 7-8 concentration points with 2-fold or 3-fold dilutions

  • Ensure the standard curve encompasses both the lower and upper limits of detection

• Blank Controls:

  • Include wells with all reagents except the sample or standard

  • Used to determine background signal and calculate the limit of detection

• Negative Controls:

  • Sample matrix without METTL25 (e.g., serum or cell lysate from METTL25 knockout models)

  • Non-specific antibody of the same isotype as the METTL25 antibody

• Positive Controls:

  • Samples with known METTL25 concentration

  • Cell lysates or tissue extracts with confirmed METTL25 expression

  • Recombinant METTL25 protein spiked into matrix similar to test samples

• Specificity Controls:

  • Competitive inhibition control: Pre-incubate detection antibody with excess METTL25 protein

  • Cross-reactivity assessment: Test against related METTL family proteins

• Technical Controls:

  • Duplicate or triplicate measurements for all samples and standards

  • Intra-assay controls: Same sample measured multiple times within one assay

  • Inter-assay controls: Same sample measured across different assay runs

• Antibody Validation Controls:

  • When using a sandwich ELISA format, verify that capture and detection antibodies recognize different epitopes

  • Test for hook effect by analyzing highly concentrated samples and their dilutions

• Data Analysis Controls:

  • Include quality control samples with known concentrations within the working range

  • Calculate coefficient of variation (CV) for replicates (aim for CV <15%)

  • Establish acceptance criteria for standard curve (r² >0.98)

Implementing these controls ensures that ELISA results for METTL25 quantification are specific, accurate, and reproducible, which is particularly important when studying this protein in experimental or clinical samples.

How can researchers differentiate between specific and non-specific binding when using METTL25 antibodies?

Distinguishing specific from non-specific binding is critical for accurate interpretation of results when using METTL25 antibodies. Several experimental approaches can help researchers make this distinction:

• Peptide Competition Assay:

  • Pre-incubate the METTL25 antibody with an excess of purified METTL25 protein or immunizing peptide

  • Apply the antibody-antigen mixture to parallel samples

  • Specific signals should be significantly reduced or eliminated, while non-specific signals will remain unchanged

  • This technique is particularly valuable for validating bands in Western blot or staining patterns in IHC/ICC

• Genetic Validation:

  • Compare antibody signal between wild-type samples and those with METTL25 knocked down (siRNA, shRNA) or knocked out (CRISPR-Cas9)

  • Specific signals should be proportionally reduced in knockdown samples or absent in knockout samples

  • Persistent signals in knockout samples indicate non-specific binding

• Multiple Antibody Approach:

  • Use two or more antibodies targeting different epitopes of METTL25

  • Consistent detection patterns across different antibodies suggest specific binding

  • Discrepancies may indicate non-specific interactions with one or more antibodies

• Isotype Control:

  • Use control antibodies of the same isotype (e.g., IgG) and host species as the METTL25 antibody

  • Apply at the same concentration as the primary antibody

  • Helps identify non-specific binding due to Fc receptor interactions or general antibody stickiness

• Titration Experiments:

  • Test a range of antibody concentrations

  • Specific binding typically shows dose-dependent signal intensity with saturation at higher concentrations

  • Non-specific binding often increases linearly with antibody concentration without reaching saturation

• Cross-Adsorption Controls:

  • Use antibodies pre-adsorbed against common cross-reactive proteins

  • Compare results with non-adsorbed antibodies

  • Reduction in background with adsorbed antibodies indicates previous non-specific interactions

• Biophysical Validation:

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) to measure binding kinetics

  • Specific antibody-antigen interactions typically show high affinity (low nM Kd) and defined on/off rates

  • Non-specific interactions often display atypical binding curves

• Immunoprecipitation Followed by Mass Spectrometry:

  • Perform IP with the METTL25 antibody and identify pulled-down proteins by mass spectrometry

  • Enrichment of METTL25 protein indicates specific binding

  • Identification of multiple unrelated proteins suggests non-specific interactions

By combining several of these approaches, researchers can confidently distinguish between specific METTL25 detection and non-specific antibody interactions, thereby increasing the reliability of their experimental results.

What are the current challenges in studying METTL25's methyltransferase activity?

Despite being classified as a putative methyltransferase , the specific biochemical activity and substrates of METTL25 remain largely uncharacterized, presenting several significant challenges for researchers:

• Substrate Identification:

  • The natural substrates (proteins, RNA, DNA, or small molecules) that METTL25 methylates remain unknown

  • Developing unbiased screening approaches to identify potential substrates requires specialized techniques like protein arrays, RNA-seq after METTL25 manipulation, or mass spectrometry-based approaches

• Enzymatic Assay Development:

  • Lack of validated biochemical assays to measure METTL25's methyltransferase activity directly

  • Need for optimization of in vitro methylation assays using recombinant METTL25 protein and radiolabeled methyl donors (e.g., S-adenosyl-L-methionine)

• Structural Characterization:

  • Limited structural information about METTL25's active site and substrate binding domains

  • Challenges in protein crystallization or cryo-EM studies of METTL25 alone or in complex with substrates and cofactors

• Cofactor Requirements:

  • Undefined cofactor dependencies beyond the presumed SAM (S-adenosyl-L-methionine) requirement

  • Potential need for protein binding partners or specific cellular conditions to activate enzymatic function

• Specificity Determination:

  • Understanding the sequence or structural determinants that confer METTL25's substrate specificity

  • Differentiating METTL25's activity from other methyltransferases in cellular contexts

• Functional Redundancy:

  • Potential overlapping functions with other METTL family members

  • Challenges in detecting phenotypes in single-gene manipulation studies due to compensatory mechanisms

• Regulation of Enzymatic Activity:

  • Unknown regulatory mechanisms controlling METTL25's activity (post-translational modifications, protein-protein interactions, subcellular localization)

  • Identifying conditions that modulate its activity in physiological or pathological contexts

• Physiological Relevance:

  • Connecting biochemical activity to cellular functions and disease relevance

  • Limited understanding of how METTL25's methyltransferase activity contributes to its observed associations with cancer

• Technical Challenges:

  • Production of active recombinant enzyme for in vitro studies

  • Development of specific inhibitors or activity probes

  • Generation of antibodies that specifically recognize methylated substrates

Addressing these challenges will require multidisciplinary approaches combining biochemistry, structural biology, cell biology, and cancer research to fully elucidate METTL25's enzymatic function and its relevance in normal physiology and disease states.

What emerging technologies might advance METTL25 research in the near future?

Several cutting-edge technologies show promise for addressing current knowledge gaps and accelerating research on METTL25:

• CRISPR-Based Functional Genomics:

  • CRISPR interference/activation (CRISPRi/a) for precise modulation of METTL25 expression

  • CRISPR screens to identify synthetic lethal interactions in cancer contexts

  • Base editing or prime editing for introducing specific mutations to study structure-function relationships

• Advanced Proteomics Methods:

  • Thermal proteome profiling (TPP) to identify proteins that interact with METTL25

  • Proximity labeling techniques (BioID, APEX) to map METTL25's protein interaction network in living cells

  • Top-down proteomics for comprehensive characterization of post-translational modifications on METTL25

• Spatial Multi-omics:

  • Spatial transcriptomics to map METTL25 expression patterns within tissue microenvironments

  • Imaging mass cytometry to correlate METTL25 expression with cellular phenotypes at single-cell resolution

  • Digital spatial profiling to analyze METTL25 in relation to other proteins in the tumor microenvironment

• Structural Biology Advances:

  • AlphaFold2 and other AI-based protein structure prediction tools to model METTL25 structure

  • Cryo-electron microscopy for structural determination of METTL25 complexes

  • Fragment-based drug discovery for developing specific METTL25 inhibitors

• Single-Cell Technologies:

  • Single-cell RNA-seq to characterize cell type-specific METTL25 expression patterns

  • Single-cell ATAC-seq to understand epigenetic regulation of METTL25

  • Single-cell proteomics to analyze METTL25 protein levels and modifications at single-cell resolution

• Methylation Detection Technologies:

  • Antibody-independent methods for detecting methylated substrates (chemical approaches)

  • Development of methylation-specific biosensors for real-time activity monitoring

  • Mass spectrometry techniques optimized for detecting and quantifying methylation sites

• Organoid and Patient-Derived Models:

  • Patient-derived organoids to study METTL25 function in physiologically relevant systems

  • Organoid-based high-throughput drug screening to identify compounds targeting METTL25-dependent pathways

  • Co-culture systems to investigate METTL25's role in cell-cell communication within the tumor microenvironment

• Live Cell Imaging Approaches:

  • FRET/BRET biosensors to monitor METTL25 activity in real time

  • Super-resolution microscopy to track METTL25 localization and dynamics

  • Optogenetic tools to precisely control METTL25 activity in specific cellular compartments

• Systems Biology Integration:

  • Multi-omics data integration to place METTL25 in broader cellular networks

  • Machine learning approaches to predict METTL25 functions and disease associations

  • Network medicine approaches to identify therapeutic opportunities related to METTL25 dysregulation

These emerging technologies and approaches have the potential to overcome current limitations in METTL25 research and provide deeper insights into its biochemical functions, regulatory mechanisms, and potential therapeutic applications in cancer and other diseases.