mat2b Antibody

What is MAT2B and why is it an important research target?

MAT2B (Methionine Adenosyltransferase 2 Subunit Beta) serves as the regulatory subunit of S-adenosylmethionine synthetase 2, an enzyme catalyzing the formation of S-adenosylmethionine from methionine and ATP . The protein regulates MAT2A catalytic activity by altering its kinetic properties, specifically increasing its affinity for L-methionine . MAT2B has gained research interest because it interacts with GIT1 to increase ERK activity and growth in human liver and colon cancer cells . Additionally, studies have shown that MAT2B expression correlates with poor prognosis in triple-negative breast cancer, making it a potentially valuable biomarker and therapeutic target .

What are the common applications for MAT2B antibodies in research?

MAT2B antibodies are primarily utilized in several laboratory techniques:

• Western Blot (WB): Commonly used at dilutions between 1:500-1:3000 to detect MAT2B protein in cell and tissue lysates . Typical observed band size is approximately 37 kDa, close to the predicted size of 38 kDa .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Typically used at dilutions of 1:100-1:1000 for visualizing cellular localization of MAT2B . This technique has been successfully demonstrated with paraformaldehyde-fixed A431 cells .

• ELISA: MAT2B can be detected in various sample types including serum, plasma, cell culture supernatant, and tissue lysates using double antibody sandwich ELISA methods .

How should MAT2B antibodies be stored and handled for optimal performance?

For optimal antibody performance, researchers should follow these handling guidelines:

• Store aliquoted antibody at -20°C or -80°C to maintain stability

• Avoid repeated freeze-thaw cycles which can compromise antibody integrity

• Most commercial antibodies are shipped with polar packs and should be stored immediately upon receipt at recommended temperatures

• Some formulations contain preservatives like 0.01% Thimerosal and are prepared in buffers such as 0.1M Tris (pH 7), 0.1M Glycine, and 20% Glycerol

• Working dilutions should be prepared fresh before use in experiments to ensure consistent results

What signaling pathways does MAT2B interact with, and how can antibodies help study these interactions?

MAT2B participates in several key signaling pathways that researchers can investigate using specific antibodies:

• MEK/ERK Pathway: MAT2B forms a complex with GIT1 that serves as a scaffold for activating MEK . Co-immunoprecipitation experiments using MAT2B antibodies have demonstrated that MAT2B and GIT1 interact with both c-Raf and B-Raf, leading to increased recruitment of these kinases to MEK1/2 . This interaction enhances ERK activity and promotes cellular growth in cancer cells .

• AKT Pathway: Research has shown that knockdown of MAT2B in triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) represses the expression of phosphorylated AKT . This suggests that MAT2B may regulate cell survival and proliferation through the AKT signaling pathway.

• ERK1/2 Pathway: Similar to its effects on AKT, MAT2B knockdown also reduces phosphorylated ERK1/2 levels in breast cancer cells . Both phosphorylated AKT and ERK1/2 inhibitors have been shown to reduce cell growth and migration while inducing apoptosis in MDA-MB-231 cells .

Researchers can use MAT2B antibodies in combination with phospho-specific antibodies targeting these pathways to explore the mechanistic relationships through techniques such as co-immunoprecipitation, Western blotting, and immunofluorescence microscopy.

How can knockout validation be used to ensure MAT2B antibody specificity?

Knockout validation represents the gold standard for confirming antibody specificity in research applications. When validating MAT2B antibodies:

• Generation of knockout models: CRISPR-Cas9 or similar gene editing techniques can be used to create MAT2B knockout cell lines (as demonstrated with 293T cells in the search results) .

• Western blot comparison: Running wild-type and MAT2B knockout cell extracts side by side on SDS-PAGE, then probing with the MAT2B antibody allows researchers to confirm specificity . A specific antibody will show the expected band in wild-type samples but no band in the knockout samples.

• Immunocytochemistry validation: Similar comparative analysis between wild-type and knockout cells can be performed using immunofluorescence staining to confirm specificity in this application.

• Quantitative analysis: Densitometry of Western blots can provide quantitative evidence of antibody specificity, with signals in knockout cells typically showing >90% reduction compared to wild-type.

This approach helps researchers avoid misleading results from non-specific antibody binding, which is particularly important when studying proteins like MAT2B that may have multiple variants (V1 and V2) .

What are the technical considerations when using MAT2B antibodies to study its role in cancer progression?

When investigating MAT2B's role in cancer using antibodies, researchers should consider:

What methodological approaches can resolve contradictory results when using MAT2B antibodies?

When researchers encounter contradictory results with MAT2B antibodies, several methodological approaches can help resolve discrepancies:

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of MAT2B to confirm findings. This helps rule out epitope-specific artifacts.

• Complementary detection methods: Combine antibody-based methods with orthogonal techniques such as:

  • mRNA expression analysis (qRT-PCR) to correlate protein levels with transcript levels

  • Mass spectrometry to confirm protein identity and modification state

  • Functional assays (RNAi knockdown or overexpression) to validate biological effects

• Standardized protocols: Ensure consistent experimental conditions across laboratories by:

  • Using validated positive and negative controls

  • Standardizing cell culture conditions, lysis methods, and antibody dilutions

  • Documenting detailed protocols for reproducibility

• Verification through knockout models: As discussed earlier, using MAT2B knockout cell lines as negative controls provides definitive validation of antibody specificity .

• Addressing post-translational modifications: Consider whether contradictory results might stem from detection of different post-translational modifications or protein-protein interactions that could mask epitopes in certain contexts.

What are the optimal conditions for using MAT2B antibodies in different experimental applications?

When optimizing these protocols, researchers should consider:

• For Western blots: SDS-PAGE gel percentage should be appropriate for the protein size (10% for MAT2B at 38 kDa)

• For immunofluorescence: Background can be minimized with proper blocking and antibody dilution; confocal microscopy has been successfully used to visualize MAT2B interactions with proteins like B-Raf and c-Raf

• For ELISA: The standard curve range for commercial kits typically spans 0.313-20ng/ml with a sensitivity of approximately 0.188ng/ml

How can MAT2B antibodies be used to study protein-protein interactions in cancer pathways?

MAT2B antibodies are valuable tools for investigating protein-protein interactions, particularly in cancer-related signaling pathways:

• Co-immunoprecipitation (Co-IP): MAT2B antibodies can pull down protein complexes, allowing identification of interacting partners. This approach has successfully demonstrated interactions between MAT2B and GIT1, as well as with c-Raf and B-Raf in both RKO and HepG2 cells .

• Proximity ligation assay (PLA): This technique can detect protein interactions in situ with high sensitivity and specificity by generating fluorescent signals only when two proteins are in close proximity (<40nm).

• Immunofluorescence colocalization: Confocal microscopy combined with MAT2B antibodies has confirmed that endogenous MAT2B interacts with B-Raf and c-Raf in HepG2 cells . This visual approach provides spatial information about where in the cell these interactions occur.

• Pull-down assays with recombinant proteins: Using purified components in an in vitro system has shown that enhanced recruitment of B-Raf and c-Raf to MEK1 occurs only when both MAT2B and GIT1 are present .

• Functional validation through mutagenesis: After identifying interaction partners, researchers can use site-directed mutagenesis to disrupt specific binding sites and assess the impact on downstream signaling, such as MEK activation.

These approaches collectively provide robust evidence for MAT2B's role as part of a signaling complex that modulates cancer cell growth through the MEK/ERK pathway.

How are MAT2B antibodies being used to explore therapeutic targeting in cancer?

MAT2B antibodies are instrumental in the development of targeted cancer therapies through several research approaches:

• Biomarker validation: MAT2B expression has been correlated with poor prognosis in triple-negative breast cancer, suggesting its potential as a prognostic biomarker . Antibodies allow researchers to assess expression levels in clinical samples and correlate with patient outcomes.

• Mechanism elucidation: Studies using MAT2B antibodies have revealed that knockdown of MAT2B inhibits cell growth and migration while inducing apoptosis through suppression of AKT and ERK pathways . This mechanistic understanding is crucial for rational drug design.

• Target validation: In vivo studies have demonstrated that MAT2B knockdown significantly decreases tumor growth rates, providing validation for MAT2B as a therapeutic target . Antibodies are essential for confirming target engagement in these models.

• Combination therapy exploration: Since MAT2B affects both AKT and ERK1/2 phosphorylation, researchers are investigating how targeting MAT2B might synergize with existing inhibitors of these pathways. Antibodies allow for monitoring of pathway modulation in response to combination treatments.

• Patient stratification research: Given the correlation between MAT2B expression and clinical outcomes, researchers are exploring whether MAT2B levels (detected via antibodies) could identify patient subgroups most likely to benefit from targeted therapies.

The development of targeted therapies against MAT2B or its interaction partners represents a promising area for cancer treatment, particularly for aggressive subtypes like TNBC where treatment options remain limited.

What are the current limitations of commercially available MAT2B antibodies and how might they be addressed?

While commercial MAT2B antibodies have enabled significant research advances, several limitations exist that researchers should consider:

• Isoform discrimination: Current antibodies may not effectively distinguish between the V1 and V2 variants of MAT2B . Development of isoform-specific antibodies targeting unique regions of each variant would address this limitation.

• Species cross-reactivity: Many antibodies are optimized for human MAT2B detection, potentially limiting their utility in animal models. Validation across multiple species would enhance versatility for translational research.

• Post-translational modification detection: Standard antibodies may not detect or distinguish between different post-translational modifications of MAT2B. Phospho-specific or other modification-specific antibodies would provide deeper insights into regulatory mechanisms.

• Quantitative accuracy: Traditional Western blot approaches offer limited quantitative precision. Development of calibrated ELISA kits with precise standard curves (like the one described with a range of 0.313-20ng/ml) can improve quantitative analysis.

• Structural constraints: Antibodies may have limited access to certain epitopes in native protein complexes. Alternative approaches like aptamers or nanobodies might offer advantages for detecting MAT2B in its native conformational state.

Addressing these limitations will require continued development of next-generation antibodies and complementary detection technologies to advance our understanding of MAT2B biology in normal and pathological states.