NTMT1 Antibody

What is NTMT1 and why is it important for research?

NTMT1, also known as METTL11A or NRMT1, is the first identified eukaryotic N-terminal methyltransferase responsible for adding methyl groups to the N-terminal of specific proteins. This enzyme plays crucial roles in regulating cell mitosis, DNA damage repair, neurogenesis, and stem cell maintenance . While this modification was first discovered four decades ago, it remained largely unexplored until the recent discovery of NTMT1 in yeast and human systems . The importance of studying NTMT1 stems from its widespread activity on both histone and non-histone proteins and its critical roles in regulating chromatin interactions, DNA repair, tRNA transport, and genome stability . Dysregulation of NTMT1 has been linked to various cancers, making it a significant target for cancer research .

What are the typical cellular and subcellular localization patterns of NTMT1?

Subcellular localization analysis using immunofluorescence has demonstrated that NTMT1 is primarily located in the nucleus and cytoplasm of cells . Specifically, in A-431 (human epidermoid carcinoma) and U2OS (human osteosarcoma) cell lines, NTMT1 shows a dual localization pattern . This dual localization correlates with NTMT1's diverse functions in both nuclear processes (such as DNA repair and chromatin interactions) and cytoplasmic processes. Researchers using NTMT1 antibodies should expect positive signals in both compartments, with potentially varying intensities depending on the cell type and physiological state being studied.

How can I verify the specificity of NTMT1 antibodies?

Verifying antibody specificity is crucial for reliable research outcomes. For NTMT1 antibodies, several validation approaches are recommended:

• Positive control testing: Use cell lines known to express high levels of NTMT1, such as HCT116 or HT29 colorectal carcinoma cells, which have been well-characterized in the literature .

• Genetic knockdown/knockout validation: Compare antibody signals between wild-type cells and cells where NTMT1 has been knocked down or knocked out using siRNA, shRNA, or CRISPR-Cas9 systems. A specific antibody should show significantly reduced or absent signal in the knockdown/knockout samples.

• Overexpression testing: Compare signals between vector control and NTMT1-overexpressing cells.

• Cross-reactivity assessment: Test the antibody against purified NTMT1 and the closely related NTMT2 protein to ensure it doesn't cross-react with similar methyltransferases.

• Western blot analysis: Look for a single band at the expected molecular weight (~25-30 kDa for human NTMT1). Multiple or unexpected bands may indicate cross-reactivity.

What are the recommended fixation and permeabilization methods for immunostaining of NTMT1?

For optimal immunostaining of NTMT1, consider the following protocols:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature works well for most applications. Alternatively, methanol fixation (100% methanol at -20°C for 10 minutes) can be used for better nuclear antigen preservation.

• Permeabilization: Since NTMT1 is present in both nucleus and cytoplasm, use 0.1-0.5% Triton X-100 in PBS for 10 minutes at room temperature to ensure adequate permeabilization of both cellular compartments.

• Antigen retrieval: For formalin-fixed paraffin-embedded (FFPE) tissues, heat-mediated antigen retrieval using citrate buffer (pH 6.0) is recommended to expose NTMT1 epitopes that may be masked during fixation.

• Blocking: Use 5% normal serum (from the species in which the secondary antibody was raised) with 0.1% Triton X-100 and 1% BSA in PBS for 1 hour at room temperature to reduce background staining.

How can NTMT1 antibodies be used to investigate cancer progression mechanisms?

NTMT1 antibodies serve as powerful tools for investigating the complex role of NTMT1 in cancer progression through multiple advanced applications:

• Expression profiling: NTMT1 antibodies can be used for immunohistochemistry (IHC) to analyze expression patterns across tumor stages and grades. This is particularly relevant as NTMT1 has been found to be significantly overexpressed in various types of tumors, including colorectal cancer, where it ranks in the top 1% of all proteins showing expression changes .

• Prognostic marker analysis: IHC scoring of NTMT1 in patient samples can help determine correlations with survival outcomes, as high levels of NTMT1 have been associated with poor survival in several cancer types .

• Mechanistic studies: NTMT1 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to identify genomic regions affected by NTMT1-mediated methylation, providing insights into its regulatory mechanisms.

• Protein-protein interaction mapping: Co-immunoprecipitation using NTMT1 antibodies can reveal interaction partners in cancer cells versus normal cells, helping to elucidate context-dependent oncogenic or tumor-suppressive functions .

• Post-translational modification analysis: NTMT1 antibodies can be used in combination with antibodies against specific N-terminally methylated proteins to investigate the relationship between NTMT1 expression and its enzymatic activity in cancer cells.

What is the relationship between NTMT1 and NTMT2, and how can antibodies help distinguish their functions?

NTMT1 and NTMT2 (also known as METTL11B) are related N-terminal methyltransferases with potentially overlapping yet distinct functions. NTMT1 antibodies play a crucial role in distinguishing between these enzymes:

• Selectivity assessment: High-quality NTMT1 antibodies that don't cross-react with NTMT2 are essential for accurate functional studies. The search results indicate that PROTAC degraders offer a way to target cellular NTMT1 specifically, which is significant for studying the relationship between NTMT1 and NTMT2 . This selectivity has been previously challenging to achieve.

• Differential expression analysis: Using specific antibodies for both NTMT1 and NTMT2 in parallel experiments allows researchers to map their distinct expression patterns across tissues and cell types.

• Functional redundancy studies: In knockdown experiments of either NTMT1 or NTMT2, antibodies can help determine if one enzyme compensates for the loss of the other by monitoring changes in expression levels.

• Substrate specificity investigation: Immunoprecipitation with NTMT1 antibodies followed by mass spectrometry can identify specific substrates of NTMT1 that may differ from NTMT2 substrates.

How can NTMT1 antibodies be utilized in investigating tumor immune microenvironment interactions?

Recent research has suggested that NTMT1 may influence the tumor immune microenvironment, making NTMT1 antibodies valuable tools in immuno-oncology research:

• Multiplex immunofluorescence: Combining NTMT1 antibodies with markers for different immune cell populations (T cells, macrophages, etc.) can reveal spatial relationships between NTMT1-expressing tumor cells and infiltrating immune cells.

• Immune correlation studies: The TISIDB database has been used to explore associations between NTMT1 expression and the abundance of tumor-infiltrating lymphocytes (TILs), immunomodulators, and chemokines . NTMT1 antibodies can be used to validate these bioinformatic findings in tissue sections.

• Response to immunotherapy: NTMT1 antibody-based IHC can help stratify patients based on NTMT1 expression levels to investigate correlations with response to immune checkpoint inhibitors.

• Immunogenic cell death signaling: Research has shown that NTMT1 degrader 1 induced overexpression of calreticulin (CALR), an immunogenic cell death (ICD) signaling protein known to elicit anti-tumor immune responses . NTMT1 antibodies can be used alongside CALR antibodies to investigate this relationship in various tumor models.

What role do NTMT1 antibodies play in evaluating new NTMT1-targeted therapeutics?

NTMT1 antibodies are essential tools in the development and validation of NTMT1-targeted therapeutics:

• Target engagement verification: For small molecule inhibitors or PROTAC degraders targeting NTMT1, antibodies can confirm successful binding to the target protein through techniques like cellular thermal shift assays (CETSA).

• Protein degradation monitoring: When evaluating PROTAC degraders like degrader 1, NTMT1 antibodies are crucial for Western blot analysis to quantify the reduction in NTMT1 protein levels. Research has shown that degrader 1 can reduce NTMT1 protein levels effectively and selectively in a time- and dose-dependent manner in colorectal carcinoma cell lines .

• Pharmacodynamic marker development: NTMT1 antibodies can be used to develop immunoassays for measuring NTMT1 levels in clinical samples, serving as pharmacodynamic markers in clinical trials of NTMT1-targeted therapies.

• On-target vs. off-target effects: When testing NTMT1 inhibitors, antibodies help distinguish between effects due to NTMT1 inhibition versus non-specific effects by correlating phenotypic changes with NTMT1 expression or activity levels.

What are the optimal conditions for using NTMT1 antibodies in Western blotting?

For reliable and reproducible Western blot results with NTMT1 antibodies, consider the following protocol optimizations:

• Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. Include N-ethylmaleimide (NEM) to preserve protein methylation states.

• Protein loading: Load 20-50 μg of total protein per lane, with HCT116 or HT29 colorectal cancer cell lysates serving as positive controls .

• Gel percentage: Use 10-12% polyacrylamide gels for optimal resolution of NTMT1 (~25-30 kDa).

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour using PVDF membrane (0.45 μm pore size).

• Blocking: 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody dilution: Typically 1:1000 to 1:2000 in 5% BSA in TBST, incubated overnight at 4°C.

• Washing: 3-5 washes with TBST, 5-10 minutes each.

• Secondary antibody: HRP-conjugated or fluorescently labeled secondary antibody at 1:5000 to 1:10000 dilution in 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Detection: Enhanced chemiluminescence (ECL) substrate for HRP-conjugated secondary antibodies or direct scanning for fluorescent secondaries.

How should researchers design time-course experiments to study NTMT1 degradation using antibodies?

Time-course experiments are essential for understanding NTMT1 dynamics and degradation kinetics. Based on published research with NTMT1 degraders, the following experimental design is recommended:

• Timepoints: Collect samples at 0, 3, 6, 12, 24, and 48 hours after treatment with degraders or other compounds. Research has shown that in HCT116 cells treated with 50 μM degrader 1, >60% NTMT1 was degraded within 3 hours and almost completely depleted within 24 hours .

• Controls: Include both vehicle controls (DMSO) and negative control compounds (e.g., inactive analogs) at each timepoint.

• Cell types: Consider testing multiple cell lines, as degradation kinetics can vary significantly between cell types. For example, NTMT1 degradation was faster in HCT116 than in HT29 cells under the same conditions .

• Concentration series: Run parallel time courses with different concentrations of the treatment compound to establish both time and dose dependencies.

• Recovery assessment: After removing the treatment compound, monitor NTMT1 re-synthesis by collecting samples at regular intervals (e.g., 6, 12, 24, 48 hours post-washout).

• Detection method: Western blotting with NTMT1 antibodies is the primary method, but consider complementing with immunofluorescence to visualize subcellular changes in NTMT1 localization during degradation.

What controls should be included when using NTMT1 antibodies for immunohistochemistry?

Proper controls are essential for reliable immunohistochemistry (IHC) with NTMT1 antibodies:

• Positive tissue controls: Include colorectal cancer tissue sections, which have been shown to have high NTMT1 expression . Breast, lung adenocarcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma tissues are also suitable positive controls based on HPA data .

• Negative tissue controls: Include kidney renal clear cell carcinoma tissues, which show significantly down-regulated NTMT1 .

• Technical negative controls:

  • Primary antibody omission: Incubate one section with antibody diluent instead of primary antibody.

  • Isotype control: Use an irrelevant antibody of the same isotype and concentration as the NTMT1 antibody.

  • Peptide competition: Pre-incubate the NTMT1 antibody with excess NTMT1 blocking peptide before application to tissue.

• Internal controls: Within the same tissue section, identify cell types known to be negative for NTMT1 to serve as internal negative controls.

• Validation with multiple antibodies: If possible, confirm staining patterns with two different NTMT1 antibodies recognizing different epitopes.

How can NTMT1 antibodies be optimized for use in chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments with NTMT1 antibodies require careful optimization for successful outcomes:

• Crosslinking conditions: Start with standard 1% formaldehyde for 10 minutes at room temperature. For NTMT1, which interacts with chromatin, consider testing dual crosslinking with 1.5 mM EGS (ethylene glycol bis(succinimidyl succinate)) for 30 minutes followed by 1% formaldehyde for 10 minutes.

• Sonication parameters: Optimize sonication to achieve chromatin fragments of 200-500 bp. Start with 10-15 cycles of 30 seconds ON/30 seconds OFF at medium intensity.

• Antibody selection: Choose ChIP-grade NTMT1 antibodies validated for this application. If unavailable, test multiple antibodies against different epitopes of NTMT1.

• Antibody amount: Typically 2-5 μg per ChIP reaction, but may require optimization.

• Pre-clearing: Pre-clear chromatin with protein A/G beads to reduce background.

• Controls:

  • Input control: Save 5-10% of chromatin before immunoprecipitation.

  • Negative control: Use IgG from the same species as the NTMT1 antibody.

  • Positive control: Include antibody against a histone mark known to be present in your cell type (e.g., H3K4me3 for active promoters).

• Washing stringency: Start with standard wash buffers and increase stringency if background is high.

• Elution and reversal of crosslinks: 65°C overnight is typical for efficient reversal.

• qPCR primers: Design primers for regions expected to be associated with NTMT1, based on known NTMT1 substrates or interacting partners.

How should researchers interpret conflicting NTMT1 antibody results across different cancer types?

When facing conflicting NTMT1 antibody results across cancer types, consider these approaches to resolve discrepancies:

• Contextualize based on literature: NTMT1 has been reported to have context-dependent roles in different cancers. In some contexts, like breast cancer, it may act as a tumor suppressor, while in cervical and colorectal cancers, it appears to function as an oncogene . These biological differences may explain seemingly conflicting antibody results.

• Antibody validation: Verify that the antibody is detecting the correct protein in each cancer type through genetic knockdown/knockout controls specific to each cell line.

• Isoform consideration: Check if different cancer types express different NTMT1 isoforms that may be detected with varying efficiency by your antibody.

• Technical validation: Confirm results using multiple detection methods (e.g., Western blot, IHC, IF) and multiple antibodies against different NTMT1 epitopes.

• Microenvironment effects: Consider whether differences in the tumor microenvironment might affect NTMT1 expression or localization.

• Post-translational modifications: Investigate whether NTMT1 itself undergoes different post-translational modifications in different cancer types that might affect antibody recognition.

• Quantification methods: Standardize quantification methods across experiments and use appropriate statistical analyses to determine if apparent differences are statistically significant.

What are common artifacts and pitfalls when using NTMT1 antibodies, and how can they be avoided?

Researchers should be aware of these common artifacts and pitfalls when working with NTMT1 antibodies:

• Cross-reactivity with NTMT2: Due to sequence similarity, some NTMT1 antibodies may cross-react with NTMT2. Validate specificity using recombinant proteins or cells with NTMT1/NTMT2 knockouts.

• Background nuclear staining: As NTMT1 has nuclear localization, distinguishing specific staining from background can be challenging. Use appropriate blocking (5% normal serum + 1% BSA) and include proper negative controls.

• Fixation artifacts: Overfixation can mask epitopes. For FFPE samples, optimize antigen retrieval; for cell lines, test multiple fixation methods (PFA vs. methanol).

• Degradation during sample preparation: NTMT1 may be subject to protease activity. Always use fresh protease inhibitors in lysis buffers and keep samples cold.

• Antibody lot-to-lot variation: Significant variation can occur between lots of the same antibody. Document lot numbers and test new lots against previous ones before conducting critical experiments.

• Insufficient controls: Always include positive controls (HCT116 or HT29 cells) and negative controls (NTMT1 knockout/knockdown cells) in experiments.

• Quantification bias: When quantifying band intensity from Western blots or IHC staining intensity, use standardized protocols and appropriate normalization (GAPDH, β-actin, or total protein staining for Western blots).

• Exposure to methylation inhibitors: Cell culture with methionine-depleted media or methyltransferase inhibitors may affect NTMT1 activity and potentially its expression or stability, confounding experimental results.

How can researchers accurately quantify NTMT1 levels using antibody-based techniques?

For accurate quantification of NTMT1 levels, consider these methodological approaches:

• Western blot quantification:

  • Use housekeeping proteins (GAPDH, β-actin) or total protein staining (Ponceau S, SYPRO Ruby) for normalization.

  • Include a standard curve of recombinant NTMT1 protein at known concentrations.

  • Use digital imaging systems with a linear dynamic range rather than film exposure.

  • Perform at least three biological replicates and appropriate statistical analyses.

• ELISA development:

  • Develop a sandwich ELISA using two antibodies recognizing different NTMT1 epitopes.

  • Include a standard curve with recombinant NTMT1 protein.

  • Optimize blocking, washing, and detection conditions for maximum sensitivity and specificity.

• Immunohistochemistry scoring:

  • Use an established scoring system such as H-score (intensity × percentage of positive cells).

  • Employ digital pathology software for unbiased quantification.

  • Have multiple pathologists score samples independently to ensure reproducibility.

  • Consider multiplex immunofluorescence for co-localization studies and more precise quantification.

• Flow cytometry:

  • Optimize fixation and permeabilization for intracellular NTMT1 detection.

  • Use isotype controls to set appropriate gates.

  • Include a negative cell line or knockout control for background determination.

  • Consider using fluorescence-minus-one (FMO) controls for accurate gating.

How should researchers analyze data from experiments using NTMT1 degraders and NTMT1 antibodies?

When analyzing data from experiments using NTMT1 degraders with antibody detection, follow these guidelines:

Comparative Analysis of NTMT1 Expression Across Cancer Types

NTMT1 Degrader Performance and Activity

What are the emerging trends in NTMT1 antibody research?

Recent advances in NTMT1 research point to several emerging trends that will shape future antibody applications:

• Development of degrader-specific antibodies: As PROTAC degraders for NTMT1 become more prevalent, antibodies specifically designed to detect degrader-bound NTMT1 or to distinguish between free and degrader-bound NTMT1 will become important.

• Single-cell analysis: NTMT1 antibodies optimized for single-cell techniques such as mass cytometry (CyTOF) or single-cell Western blotting will enable more detailed analysis of NTMT1 expression heterogeneity within tumors.

• Spatial biology applications: NTMT1 antibodies validated for spatial transcriptomics and proteomics platforms will help elucidate the spatial context of NTMT1 function in complex tissues.

• Post-translational modification-specific antibodies: Development of antibodies that specifically recognize N-terminally methylated proteins will complement NTMT1 antibodies to provide a more complete understanding of NTMT1 activity.

• In vivo imaging applications: NTMT1 antibodies labeled for PET or optical imaging could enable non-invasive monitoring of NTMT1 expression in preclinical models.

How might NTMT1 antibody research contribute to future therapeutic developments?

NTMT1 antibody research stands to make significant contributions to therapeutic development in several ways:

• Patient stratification: NTMT1 antibody-based immunohistochemistry could help identify cancer patients most likely to respond to NTMT1-targeted therapies, as high NTMT1 expression is associated with poor prognosis in multiple cancer types .

• Pharmacodynamic biomarkers: NTMT1 antibodies will serve as essential tools for measuring target engagement and efficacy of NTMT1 inhibitors or degraders in clinical trials.

• Combination therapy identification: By using NTMT1 antibodies to study the effects of NTMT1 inhibition on various signaling pathways, researchers may identify synergistic drug combinations.

• Immunotherapy connections: Further investigation of the relationship between NTMT1 and immunogenic cell death signals like calreticulin using antibody-based approaches could reveal new strategies for enhancing immunotherapy efficacy.

• Developmental therapeutics: As our understanding of context-dependent NTMT1 functions grows through antibody-based research, more sophisticated therapeutic approaches may emerge that selectively target NTMT1 in specific cellular contexts or disease states.