METTL7A Antibody

What is METTL7A and what are its primary cellular functions?

METTL7A (Methyltransferase-like protein 7A) functions as a thiol S-methyltransferase that catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to alkyl and phenolic thiol-containing acceptor substrates. Along with METTL7B, it accounts for most S-thiol methylation activity in the endoplasmic reticulum of hepatocytes .

METTL7A has several key functions:

• Methylates the N6 position of adenosine residues in long non-coding RNAs (lncRNAs)

• Facilitates lncRNAs transfer into exosomes at the tumor-stroma interface

• Promotes osteogenic and odontogenic differentiation by regulating genes involved in stem cell differentiation and survival

• Can be targeted from the endoplasmic reticulum to lipid droplets

• May play a role in the assembly and release stages of hepatitis C virus (HCV)

What types of METTL7A antibodies are commonly available for research?

Based on current research resources, METTL7A antibodies are primarily available as rabbit polyclonal antibodies. Several validated options include:

• Rabbit Polyclonal antibodies that react with human samples (ab79207), suitable for Western Blot applications

• Rabbit Polyclonal antibodies that react with human, mouse, and rat samples (17092-1-AP), suitable for Western Blot, Immunohistochemistry, and ELISA applications

• Rabbit IgG antibodies that react with human and mouse samples (A14279), suitable for ELISA and Western Blot applications

These antibodies have been raised against different immunogens, including synthetic peptides within the human METTL7A protein.

What is the molecular weight and structure of METTL7A protein?

METTL7A has a calculated molecular weight of approximately 28 kDa, though observed molecular weights in experimental settings may vary:

• The calculated molecular weight is consistently reported as ~28 kDa (precisely 28,319 Da)

• Observed molecular weight in Western Blot applications is typically 25-28 kDa

• Some antibodies report an observed molecular weight of 68 kDa, which may represent a different isoform or post-translationally modified version of the protein

At least two isoforms of METTL7A are known to exist, which may explain some of the variability in observed molecular weights across different experimental conditions .

What are the optimal conditions for using METTL7A antibodies in Western Blotting?

For optimal Western Blot detection of METTL7A, researchers should follow these methodological guidelines:

• Antibody dilution ranges:

  • For antibody 17092-1-AP, use dilutions between 1:5000-1:50000

  • Titration is recommended in each specific testing system to obtain optimal results

• Sample preparation:

  • METTL7A has been successfully detected in various cell lines including HepG2 and HeLa cells

  • Tissue samples that yield positive results include mouse and rat lung tissue, and mouse liver tissue

• Expected band size:

  • Look for bands at 25-28 kDa, which is the typical observed molecular weight

  • Be aware that some antibodies may detect bands at higher molecular weights (~68 kDa)

• Storage conditions:

  • Store antibodies at -20°C where they remain stable for one year after shipment

  • Some antibodies can be stored at 4°C for up to three months

  • Avoid repeated freeze-thaw cycles

How can METTL7A antibodies be optimized for immunohistochemistry applications?

For immunohistochemistry (IHC) applications using METTL7A antibodies:

• Recommended dilution:

  • For antibody 17092-1-AP, use dilutions between 1:200-1:800

• Antigen retrieval methods:

  • Primary recommendation: Use TE buffer at pH 9.0

  • Alternative method: Use citrate buffer at pH 6.0

• Positive control tissues:

  • Human liver cancer tissue

  • Human thyroid cancer tissue

• Detection systems:

  • Standard avidin-biotin or polymer-based detection systems compatible with rabbit primary antibodies are appropriate

  • Specific detection protocols may vary by antibody and should be optimized for your specific experimental system

How can I validate the specificity of METTL7A antibodies?

Validating antibody specificity is crucial for reliable experimental results. For METTL7A antibodies, consider these validation approaches:

• Genetic validation:

  • Use CRISPR-Cas9 mediated knockout of METTL7A as a negative control

  • Research has demonstrated the utility of this approach, with complete knockout clones (e.g., 7A KO B1 and 7A KO B3) showing no METTL7A expression by Western blot

• Partial knockdown controls:

  • Include partial knockdown samples (e.g., 7A KO B4 with reduced but not eliminated expression) to demonstrate antibody sensitivity to expression levels

• Cross-reactivity assessment:

  • METTL7A antibodies are predicted not to cross-react with the homologous protein METTL7B, but this should be verified experimentally in your system

  • If studying both METTL7A and METTL7B, use specific antibodies for each protein

• Blocking peptide validation:

  • Use synthetic peptides corresponding to the immunogen to pre-absorb the antibody and confirm signal specificity

How is METTL7A involved in cancer drug resistance mechanisms?

METTL7A has emerged as a key player in resistance to histone deacetylase inhibitors (HDACis), an important class of epigenetic therapies used in cancer treatment:

• Resistance to thiol-containing HDACis:

  • METTL7A overexpression confers resistance to romidepsin and other HDACis that contain a thiol as the zinc-binding moiety

  • The methyltransferase activity of METTL7A is believed to inactivate these drugs by methylating the thiol group, preventing coordination with zinc in the HDAC binding pocket

• Experimental evidence:

  • MCF-7 DpVp300 cells (romidepsin-resistant) exhibit high METTL7A expression and cross-resistance to other thiol-based HDACis including largazole, OKI-005, KD5170, and NCH-51

  • CRISPR-Cas9 knockout of METTL7A in resistant cells restores sensitivity to thiol-based HDACis

  • In resistant cells with high METTL7A expression, treatment with HDACis fails to induce histone acetylation and p21 expression, which are restored upon METTL7A knockout

• Clinical implications:

  • METTL7A represents a previously unknown mechanism of resistance to thiol-based HDACis

  • Targeting METTL7A could potentially improve HDACi-based therapies in cancer patients

  • Methyltransferase inhibitors like DCMB can block METTL7A-mediated resistance

What is the relationship between METTL7A and METTL7B in methyltransferase activity?

METTL7A and METTL7B are homologous methyltransferases with overlapping but distinct functions:

• Functional similarities:

  • Both METTL7A and METTL7B can confer resistance to thiol-containing HDACis

  • Both proteins likely function as thiol methyltransferases

• Functional differences:

  • METTL7B confers resistance to thiol-based HDACis with less efficiency than METTL7A

  • The proteins may have different substrate specificities or cellular localizations

• Research considerations:

  • When studying methyltransferase activity in drug resistance, both proteins should be considered

  • Specific antibodies that do not cross-react between these homologs are essential for distinguishing their individual contributions

  • The combined activity of both proteins may be relevant in certain cellular contexts

How does METTL7A contribute to cell differentiation and tissue development?

METTL7A has been implicated in promoting cellular differentiation processes that are critical for tissue development:

• Osteogenic differentiation:

  • METTL7A promotes osteogenic differentiation by regulating the expression of genes involved in stem cell differentiation and survival

  • This makes it a potential factor in bone development and regeneration

• Odontogenic differentiation:

  • Similar regulatory functions have been observed in odontogenic differentiation

  • This suggests a role in tooth development and dental tissue regeneration

• Metabolic stress response:

  • METTL7A promotes cell survival and osteogenic differentiation under metabolic stress conditions

  • This adaptive response may be important for tissue development in suboptimal conditions

• Research implications:

  • METTL7A antibodies can be valuable tools for studying developmental processes

  • Tracking METTL7A expression levels during differentiation could provide insights into the temporal regulation of tissue development

  • Manipulating METTL7A expression may offer avenues for enhancing differentiation in tissue engineering applications

What is the role of METTL7A in RNA modification and exosome packaging?

METTL7A exhibits RNA methylation activity with potential implications for intercellular communication:

• lncRNA methylation:

  • METTL7A can methylate the N6 position of adenosine residues in long non-coding RNAs (lncRNAs)

  • This may alter lncRNA structure, stability, or interactions with other cellular components

• Exosome packaging:

  • METTL7A may facilitate the transfer of lncRNAs into exosomes at the tumor-stroma interface

  • This suggests a role in intercellular communication, particularly in the tumor microenvironment

• Research applications:

  • METTL7A antibodies can be used to study the localization and interactions of this protein in the context of RNA processing and exosome biogenesis

  • Co-localization studies with exosomal markers could reveal the spatial relationship between METTL7A and exosome formation sites

  • Immunoprecipitation with METTL7A antibodies may help identify associated RNAs and proteins involved in exosome packaging

What are common issues when working with METTL7A antibodies and how can they be resolved?

Researchers may encounter several challenges when working with METTL7A antibodies:

• Variable molecular weight detection:

  • Issue: Different observed molecular weights (25-28 kDa vs. 68 kDa) reported across antibodies

  • Solution: Run positive controls with known METTL7A expression to identify the correct band; consider using multiple antibodies targeting different epitopes to confirm results

• Cross-reactivity concerns:

  • Issue: Potential cross-reactivity with METTL7B due to sequence similarity

  • Solution: Verify antibody specificity using METTL7A knockout controls; use antibodies specifically tested for lack of cross-reactivity with METTL7B

• Optimization for different applications:

  • Issue: An antibody that works well for Western blot may not perform optimally for immunohistochemistry

  • Solution: Each application requires separate optimization; follow recommended dilution ranges for specific applications (WB: 1:5000-1:50000; IHC: 1:200-1:800)

• Tissue-specific expression variations:

  • Issue: METTL7A expression levels vary across tissues, affecting detection sensitivity

  • Solution: Include positive control samples with known high METTL7A expression (e.g., liver tissue, HepG2 cells)

How can I design experiments to investigate METTL7A's role in drug resistance mechanisms?

To study METTL7A's involvement in drug resistance, particularly to thiol-containing HDACis, consider these experimental approaches:

• Expression analysis in resistant vs. sensitive cells:

  • Compare METTL7A protein levels in drug-sensitive parental cells vs. resistant derivatives using validated antibodies

  • Examples: MCF-7 vs. MCF-7 DpVp300 cells; HuT78 vs. HuT78 DpVp50 and HuT78 DpP75 cells

• Genetic manipulation strategies:

  • Knockout: Generate METTL7A knockout cells using CRISPR-Cas9 to assess the impact on drug sensitivity

  • Overexpression: Introduce METTL7A into sensitive cells to determine if this is sufficient to confer resistance

  • Partial knockdown: Create cells with reduced but not eliminated expression to evaluate dose-dependent effects

• Functional readouts:

  • Cell viability assays with thiol-containing HDACis (romidepsin, largazole, OKI-005, KD5170, NCH-51)

  • Western blot analysis of histone acetylation and p21 expression as markers of HDAC inhibition

  • Cell cycle analysis to assess biological consequences of drug treatment

• Methyltransferase activity assays:

  • In vitro methylation assays using recombinant METTL7A and thiol-containing HDACis as substrates

  • Use methyltransferase inhibitors like DCMB to block METTL7A activity and potentially restore drug sensitivity

What are emerging applications for METTL7A antibodies in cancer research?

METTL7A antibodies are becoming increasingly valuable tools in cancer research, with several promising applications:

• Biomarker development:

  • METTL7A expression may serve as a predictive biomarker for response to HDACi therapy in cancer patients

  • Immunohistochemistry using validated METTL7A antibodies could help stratify patients for clinical trials

• Drug resistance mechanisms:

  • METTL7A antibodies can facilitate investigation of resistance mechanisms to thiol-containing HDACis

  • Understanding the relationship between METTL7A expression and drug efficacy could inform combination therapy strategies

• Tumor-stroma interactions:

  • Given METTL7A's role in facilitating lncRNA transfer into exosomes at the tumor-stroma interface, antibodies can help map these interactions

  • This may reveal new insights into how cancer cells communicate with their microenvironment

• Therapeutic target validation:

  • As a potential mediator of drug resistance, METTL7A itself may become a therapeutic target

  • Antibodies can be used to validate knockdown efficiency in preclinical models and assess impact on tumor growth and drug sensitivity

How might METTL7A research contribute to understanding cellular metabolism and lipid biology?

METTL7A has connections to cellular metabolism and lipid biology that warrant further investigation:

• Lipid droplet biology:

  • METTL7A can be targeted from the endoplasmic reticulum to lipid droplets, where it recruits cellular proteins to form functional organelles

  • Antibodies can help track this localization and identify associated proteins

• Metabolic stress adaptation:

  • METTL7A promotes cell survival under metabolic stress conditions

  • Understanding this mechanism could reveal new aspects of cellular adaptation to metabolic challenges

• Liver metabolism:

  • METTL7A accounts for significant S-thiol methylation activity in hepatocyte endoplasmic reticulum

  • This suggests important roles in liver metabolism that could be explored using tissue-specific antibody studies

• Methylation of metabolic intermediates:

  • As a methyltransferase, METTL7A may modify metabolic intermediates, potentially affecting metabolic fluxes

  • Antibodies could help identify the cellular contexts where this activity is most relevant