MAT2A Antibody

What is MAT2A and why is it important in research?

MAT2A (methionine adenosyltransferase II, alpha) is a critical enzyme that catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP . As the principal biological methyl donor in cells, SAM plays an essential role in methylation processes including epigenetic regulation. MAT2A has significant relevance in cancer research, particularly in tumors with co-deletion of p16 and MTAP genes which have shown sensitivity to MAT2A inhibition . The protein has a molecular weight of approximately 43.7 kDa (canonical form) and consists of 395 amino acid residues . Understanding MAT2A function and distribution is crucial for multiple fields including oncology, metabolism research, and epigenetics.

How does MAT2A differ from MAT1A and what are the implications for experimental design?

MAT1A and MAT2A are encoded by different genes but catalyze the same reaction forming SAM. The key differences affect experimental design considerations:

• Expression patterns: MAT1A is predominantly expressed in liver, while MAT2A is expressed in extrahepatic tissues and in fetal liver .

• Subcellular localization: While MAT1A is primarily cytoplasmic, MAT2A shows both cytoplasmic and nuclear immunoreactivity, which has important implications for cancer research .

• Functional differences: Studies should consider that replacing MAT1A with MAT2A (a switch observed in liver cancer) correlates with more aggressive growth and lower SAM levels.

When designing experiments, researchers should select antibodies specific to either MAT1A or MAT2A to avoid cross-reactivity and clearly establish which isoform they are investigating. The subcellular distribution should be carefully evaluated using fractionation techniques or immunofluorescence imaging.

What are the optimal dilution ratios for different applications of MAT2A antibody?

The optimal dilution ratios for MAT2A antibody applications vary based on the specific technique:

These ranges should be considered starting points . Each antibody lot and experimental system may require optimization. It is recommended to perform a titration series with your specific samples to determine the optimal antibody concentration that maximizes specific signal while minimizing background. For reproducibility, maintain consistent antibody dilutions across experimental replicates.

How should sample preparation differ for detecting nuclear versus cytoplasmic MAT2A?

Given that MAT2A shows both cytoplasmic and nuclear localization, proper sample preparation is critical:

• For nuclear fraction isolation: Use a nuclear extraction protocol with appropriate nuclear lysis buffers containing DNase. Confirm fraction purity by probing for nuclear-specific markers (e.g., Lamin B) alongside cytoplasmic markers (e.g., GAPDH).

• For cytoplasmic fraction: Employ gentle lysis conditions that preserve the nuclear membrane integrity. After removing nuclei by centrifugation, confirm absence of nuclear contamination.

• For calculating C/N ratio: Perform quantitative immunofluorescence or subcellular fractionation followed by Western blot. For IF, nuclei should be counterstained with DAPI, and fluorescence intensity should be measured in defined nuclear and cytoplasmic regions .

• Fixation considerations: For preserved localization in immunofluorescence studies, paraformaldehyde fixation (4%) for 15-20 minutes is generally effective. Over-fixation may mask epitopes, particularly for nuclear proteins.

How can MAT2A antibodies be used to evaluate the relationship between MAT2A localization and cancer progression?

The subcellular localization of MAT2A has been shown to have prognostic significance, particularly in breast cancer. Research methodologies to investigate this relationship include:

The methodology should include rigorous quantification of staining intensity in both compartments using image analysis software and appropriate statistical analysis of the resulting ratios in relation to clinical outcomes.

What strategies should be employed when using MAT2A antibodies to study the effects of MAT2A inhibitors?

When using MAT2A antibodies to evaluate MAT2A inhibitors (like compound 28 mentioned in search result ), consider these methodological approaches:

• Protein expression and localization changes:

  • Monitor changes in MAT2A expression levels and subcellular distribution using Western blot and immunofluorescence before and after inhibitor treatment.

  • Use fractionation to isolate cytoplasmic and nuclear components separately to detect compartment-specific changes.

• Target engagement verification:

  • Develop thermal shift assays using MAT2A antibodies to confirm direct binding of inhibitors to MAT2A protein.

  • Consider combining with CETSA (Cellular Thermal Shift Assay) methodology for in-cell confirmation.

• Functional readouts:

  • Measure SAM levels as a direct readout of MAT2A activity alongside antibody-based detection of the protein itself.

  • Assess downstream methylation events (e.g., histone methylation) through specific antibodies against methylated targets.

• Resistance mechanisms:

  • Use MAT2A antibodies to identify potential compensatory increases in expression following prolonged inhibitor treatment.

  • Investigate binding partners through co-immunoprecipitation with MAT2A antibodies before and after inhibitor treatment.

• In vivo modeling:

  • For xenograft studies, use IHC with MAT2A antibodies to assess changes in distribution patterns following inhibitor administration .

This comprehensive approach enables evaluation of both direct target engagement and downstream functional consequences of MAT2A inhibition.

What are common pitfalls in detecting MAT2A in IHC and how can they be resolved?

Several challenges can arise when detecting MAT2A in immunohistochemistry:

• Epitope masking issues:

  • Problem: Inadequate antigen retrieval, particularly for formalin-fixed paraffin-embedded (FFPE) tissues.

  • Solution: Optimize antigen retrieval conditions. Based on research data, TE buffer at pH 9.0 is suggested for MAT2A, although citrate buffer at pH 6.0 may be used as an alternative .

• Signal specificity concerns:

  • Problem: Difficulty distinguishing between MAT2A and MAT1A due to structural similarity.

  • Solution: Validate antibody specificity using positive and negative control tissues. Consider including liver tissue (high in MAT1A) vs. extrahepatic tissues (predominant MAT2A expression) as cross-validation.

• Subcellular localization accuracy:

  • Problem: Inconsistent or unclear nuclear vs. cytoplasmic staining.

  • Solution: Use high-resolution microscopy with appropriate counterstains for nuclei. For accurate C/N ratio quantification, employ digital image analysis with software capable of nuclear/cytoplasmic segmentation.

• Reproducibility issues:

  • Problem: Variability between experiments or samples.

  • Solution: Standardize fixation time, section thickness (4-5 μm recommended), and staining protocols. Include positive controls with established staining patterns in each run.

• Background staining:

  • Problem: High background obscuring specific MAT2A signal.

  • Solution: Increase blocking time (1-2 hours with 5% normal serum from the species of secondary antibody), optimize antibody dilution (starting with 1:50-1:500 range), and consider adding 0.1-0.3% Triton X-100 for better penetration .

How can researchers validate the specificity of MAT2A antibodies in their experimental systems?

Rigorous validation of MAT2A antibodies is essential for reliable research outcomes:

• Genetic validation approaches:

  • Generate MAT2A knockout or knockdown models (CRISPR-Cas9 or siRNA) and confirm loss of signal with the antibody.

  • Use overexpression systems to confirm increased signal detection in proportion to expression levels.

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide (when available) prior to immunostaining.

  • Signal should be significantly reduced if the antibody is specific.

• Multi-antibody comparison:

  • Test multiple antibodies targeting different epitopes of MAT2A.

  • Consistent localization patterns across antibodies increases confidence in specificity.

• Multi-application concordance:

  • Compare results across different techniques (WB, IF, IHC, IP) using the same antibody.

  • Consistent molecular weight detection and subcellular localization patterns support specificity.

• Known expression pattern verification:

  • Test the antibody in tissues/cells with established MAT2A expression patterns.

  • For example, validate differential detection between HepG2 cells (known to express MAT2A) and other cell types with varying expression levels .

• Mass spectrometry confirmation:

  • Perform immunoprecipitation with the MAT2A antibody followed by mass spectrometry to confirm the identity of the pulled-down proteins.

How can MAT2A antibodies be used to investigate the relationship between methionine metabolism and cancer progression?

MAT2A antibodies offer valuable tools for exploring methionine metabolism's role in cancer:

• Metabolic profiling correlations:

  • Combine MAT2A protein quantification (via Western blot) with metabolomic analyses of SAM/SAH ratios.

  • Correlate changes in MAT2A levels with alterations in one-carbon metabolism and methylation status.

• Multi-omics approach:

  • Integrate MAT2A antibody-based protein detection with transcriptomic data on methionine cycle enzymes.

  • Correlate MAT2A protein levels and localization with global DNA methylation patterns and histone methylation marks.

• Tumor microenvironment studies:

  • Use multiplexed immunofluorescence to examine MAT2A expression in different cell populations within the tumor microenvironment.

  • Investigate how stromal/immune cell MAT2A expression patterns differ from tumor cells.

• Therapeutic response prediction:

  • Apply MAT2A antibodies to patient-derived samples to determine if expression levels or C/N ratio can predict response to methionine-restriction therapies or MAT2A inhibitors.

  • Develop IHC scoring systems based on MAT2A C/N ratio for potential clinical application.

• Cell cycle coordination:

  • Use MAT2A antibodies alongside cell cycle markers to track the relationship between MAT2A localization and cell cycle progression.

  • Previous research has indicated that MAT2A nuclear translocation occurs after the G1/S checkpoint, enabling epigenetic histone methylation during DNA replication .

This integrated approach can help elucidate how methionine metabolism dysregulation contributes to cancer development and identify potential intervention points.

What methodological considerations are important when studying MAT2A in MTAP-deleted cancers?

MTAP (methylthioadenosine phosphorylase) deletion creates a metabolic vulnerability that can be targeted through MAT2A inhibition . When studying MAT2A in MTAP-deleted cancers:

• MTAP status verification:

  • Confirm MTAP deletion status in cell lines or patient samples using paired antibodies against both MTAP and MAT2A.

  • Western blot or IHC should be used to confirm protein-level absence of MTAP in addition to genomic deletion confirmation.

• Synthetic lethality assessment:

  • When testing MAT2A inhibitors, compare efficacy between isogenic cell lines with and without MTAP deletion.

  • Use MAT2A antibodies to confirm that protein levels are comparable between lines to establish that differential sensitivity is not due to variable MAT2A expression.

• Metabolite measurement correlation:

  • Correlate MAT2A protein levels with measurements of relevant metabolites (MTA, SAM, methionine).

  • Consider how changes in these metabolites affect MAT2A expression or localization using appropriate antibody-based detection methods.

• Xenograft models:

  • When establishing MTAP-knockout xenograft models (like the HCT116 MTAP KO model mentioned in result ), use IHC with MAT2A antibodies to confirm maintained expression in the in vivo setting.

  • Monitor changes in MAT2A expression or localization during treatment with inhibitors.

• Combination therapy investigations:

  • When studying MAT2A inhibitors in combination with other agents, use antibody-based methods to track changes in MAT2A expression, stability, or post-translational modifications that might indicate adaptation.

These methodological considerations ensure that the specific context of MTAP deletion is properly accounted for when investigating MAT2A as a therapeutic target.

How can researchers use MAT2A antibodies to investigate post-translational modifications of MAT2A?

Investigating post-translational modifications (PTMs) of MAT2A requires specialized approaches:

• Combined antibody strategies:

  • Use general MAT2A antibodies for immunoprecipitation followed by PTM-specific antibodies (phospho, acetyl, ubiquitin, etc.) for detection.

  • Alternatively, use PTM-specific antibodies for IP followed by MAT2A antibody detection.

• Modification-specific protocols:

  • For phosphorylation studies: Add phosphatase inhibitors to all buffers during sample preparation.

  • For acetylation studies: Include deacetylase inhibitors (e.g., TSA, nicotinamide).

  • For ubiquitination: Add deubiquitinase inhibitors (e.g., NEM, iodoacetamide).

• Mass spectrometry validation:

  • Perform IP with MAT2A antibodies, followed by mass spectrometry to identify specific modification sites.

  • Use this information to develop or select site-specific modification antibodies when available.

• Functional correlations:

  • Investigate how PTMs affect MAT2A subcellular localization by correlating modification status with C/N ratio.

  • Determine if PTMs change during cell cycle progression or in response to cellular stressors.

• Inhibitor response studies:

  • Monitor changes in PTM patterns before and after treatment with MAT2A inhibitors to identify potential feedback mechanisms.

This approach enables deeper understanding of MAT2A regulation beyond mere expression levels.

What approaches should be used when investigating the interactions between MAT2A and MAT2B using antibody-based methods?

MAT2A functionality is influenced by its regulatory subunit MAT2B. To study their interactions:

• Co-immunoprecipitation optimization:

  • Use MAT2A antibodies for IP followed by MAT2B antibody detection or vice versa.

  • Optimize lysis conditions to preserve protein-protein interactions (mild detergents like 0.5% NP-40 or CHAPS rather than harsh detergents like SDS).

  • Include appropriate controls (IgG control IP, input samples).

• Proximity ligation assay (PLA):

  • Apply in situ PLA to visualize and quantify MAT2A-MAT2B interactions within cells.

  • This method provides spatial information about where in the cell these interactions predominantly occur.

• FRET/BRET analyses:

  • While not directly antibody-based, these techniques can complement antibody studies.

  • Use findings from antibody-based co-localization to guide the design of FRET experiments examining MAT2A-MAT2B interactions in live cells.

• Mammalian two-hybrid systems:

  • Validate interactions identified through antibody-based methods using orthogonal techniques.

• Competition studies:

  • Use antibodies that target known interaction domains to determine if they disrupt MAT2A-MAT2B binding.

  • This approach can identify critical regions for the interaction.

• Sequential immunoprecipitation:

  • Perform tandem IP experiments to isolate MAT2A-MAT2B complexes and identify additional binding partners.

These methodological approaches provide comprehensive insights into the regulatory interactions between MAT2A and its binding partners.

What are the critical factors for achieving consistent MAT2A antibody results across different experimental batches?

Ensuring reproducibility in MAT2A antibody experiments requires attention to multiple factors:

• Antibody selection and storage:

  • Choose antibodies with validated specificity for MAT2A (e.g., those validated in KD/KO studies).

  • Maintain proper storage conditions (-20°C or -80°C as recommended) and avoid repeated freeze-thaw cycles (aliquot upon receipt).

  • Record and use consistent antibody lots when possible; if changing lots, perform side-by-side validation.

• Sample preparation standardization:

  • Standardize cell harvesting conditions (confluence, passage number).

  • Use consistent lysis buffers and protocols (consider that nuclear MAT2A may require specific extraction methods).

  • Maintain protein sample stability with appropriate protease/phosphatase inhibitors.

• Protocol documentation and execution:

  • Create detailed protocols including exact buffer compositions, incubation times, and temperatures.

  • For IHC/IF, standardize fixation conditions, antigen retrieval methods (TE buffer pH 9.0 recommended for MAT2A) , and blocking solutions.

  • Maintain consistent antibody dilutions (e.g., 1:1000-1:4000 for WB; 1:50-1:500 for IHC) .

• Controls implementation:

  • Include positive controls (cell lines with known MAT2A expression such as HEK-293, HeLa, HepG2) .

  • Use negative controls (primary antibody omission, non-specific IgG, ideally MAT2A-depleted samples).

  • Consider internal loading controls appropriate for the cellular compartment being studied.

• Image acquisition standardization:

  • Use consistent exposure settings for microscopy or chemiluminescence detection.

  • Implement standard quantification methods for C/N ratio calculations.

These practices minimize variability and enhance reproducibility across experiments.

How should researchers adjust protocols when using MAT2A antibodies across different species?

When adapting MAT2A antibody protocols across species:

• Epitope conservation analysis:

  • Check sequence homology of the immunogen peptide across target species.

  • MAT2A is relatively conserved, with antibodies often recognizing human, mouse, and rat orthologs, but verification is essential .

• Species-specific validation:

  • Test antibody reactivity in positive control samples from each species.

  • Confirm specificity through knockdown experiments in cell lines from the target species.

  • Verify expected molecular weight (may vary slightly between species).

• Protocol optimization by species:

  • Adjust antibody concentrations based on epitope conservation (less conservation may require higher concentrations).

  • Modify antigen retrieval methods for FFPE tissues from different species (generally more aggressive retrieval for less conserved epitopes).

  • Adapt blocking solutions to use serum from the species of the secondary antibody.

• Tissue-specific considerations:

  • Account for potential differences in MAT2A expression patterns between species.

  • Consider natural variation in subcellular distribution when interpreting C/N ratios across species.

• Cross-reactivity testing:

  • Evaluate potential cross-reactivity with MAT1A, which shares structural similarity with MAT2A.

  • This is particularly important in liver samples where MAT1A expression is high.

These adjustments ensure valid cross-species comparisons when studying MAT2A biology.