MOGAT2 Antibody

What is MOGAT2 and what are its biological functions?

MOGAT2 (also known as MGAT2) is an enzyme that catalyzes the synthesis of diacylglycerol from 2-monoacylglycerol and fatty acyl-CoA. The enzyme plays a central role in the absorption of dietary fat in the small intestine by catalyzing the resynthesis of triacylglycerol in enterocytes. MOGAT2 forms a complex with diacylglycerol O-acyltransferase 2 in the endoplasmic reticulum, and this complex catalyzes the synthesis of triacylglycerol . The protein has a preference toward monoacylglycerols containing unsaturated fatty acids in the order of C18:3 > C18:2 > C18:1 > C18:0 at the sn-2 position . MOGAT2 can also use 1-monoalkylglycerol as an acyl acceptor for the synthesis of monoalkyl-monoacylglycerol and subsequently may add another acyl chain producing monoalkyl-diacylglycerol, although with lower efficiency .

What types of MOGAT2 antibodies are available for research?

Several types of MOGAT2 antibodies are available for research applications:

Most available MOGAT2 antibodies are rabbit polyclonal antibodies with various immunogens, including E.coli-derived human recombinant protein (amino acids M1-C334) and peptide-based immunogens . The antibodies can recognize all three isoforms of MOGAT2 and typically have an observed molecular weight of 36-38 kDa .

What are the recommended storage conditions for MOGAT2 antibodies?

For optimal antibody performance, most MOGAT2 antibodies should be stored at -20°C and remain stable for one year after shipment . Some antibodies are provided in storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . It's important to note that aliquoting is generally unnecessary for -20°C storage, but following manufacturer-specific recommendations is advised. Some products (20μl sizes) may contain 0.1% BSA . Avoid repeated freeze-thaw cycles which can lead to loss of antibody activity and increased background signal.

What are the optimal applications and dilutions for MOGAT2 antibodies?

MOGAT2 antibodies can be utilized in multiple applications with specific recommended dilutions:

It is recommended that researchers titrate each antibody in their specific testing systems to obtain optimal results, as optimal dilutions may be sample-dependent .

How can I validate the specificity of a MOGAT2 antibody?

Validating antibody specificity is crucial for reliable experimental results. For MOGAT2 antibodies, consider the following approaches:

• Positive controls: Use tissues/cells known to express MOGAT2 (mouse small intestine tissue, HEK-293 cells, rat small intestine)

• Negative controls: Include samples with known low or absent MOGAT2 expression

• Genetic controls: Utilize MOGAT2 knockout or knockdown samples where available. Mogat2-/- mice are commercially available and can provide valuable negative control tissues

• Multiple antibody validation: Compare results using antibodies targeting different epitopes of MOGAT2

• Molecular weight verification: Confirm the detected protein corresponds to the expected molecular weight (36-38 kDa for MOGAT2)

• Cross-reactivity testing: Ensure the antibody doesn't recognize related proteins (like DGAT1, DGAT2) by using purified proteins or overexpression systems

• Immunogen competition: Pre-incubate the antibody with the immunizing peptide or protein to confirm signal specificity

How can I develop a cell-based assay for MOGAT2 inhibitor screening?

A cell-based assay for MOGAT2 inhibitor screening can be developed using the following methodology:

• Cell line selection: Use murine secretin tumor cell-1 line of enteroendocrine origin to construct human MOGAT2-expressing recombinant cell lines

• Assay platform: Implement a high-resolution LC/MS platform instead of traditional TLC techniques to avoid low throughput and hazardous radiolabeled substrates

• Substrate selection: Utilize stable isotope-labeled D31-palmitate to selectively trace cellular diacylglycerol (DAG) synthesis activity

• Detection method: Monitor incorporation of stable isotope-labeled substrate into DAG using LC/MS, which dramatically reduces background interference and increases sensitivity compared to traditional methods

• Controls: Include positive controls (known MOGAT2 inhibitors) and negative controls to validate assay performance

• Inhibitor evaluation: Test candidate inhibitors from different chemotypes to characterize their effects on MOGAT2 activity

This approach provides a robust methodology for screening, developing, and evaluating MOGAT2 inhibitors with potential applications in addressing obesity and related disorders .

How do I distinguish MOGAT2 activity from other acyltransferases in cellular assays?

Distinguishing MOGAT2 activity from other acyltransferases presents significant methodological challenges:

• Multiple enzymes with MGAT activity: In hepatic cell lines like HepG2, DGAT1 accounts for approximately 90% of in vitro MGAT activity while showing the ability to use 2-monoacylglycerol as a substrate

• Selective inhibition approach: Use selective inhibitors to parse contributions:

  • MGAT2 inhibitors reduce MGAT2 activity by ~80% with negligible effects on MGAT3 and DGAT1

  • MGAT3 inhibitors selectively target MGAT3 activity

  • Combined inhibition of MGAT2 and MGAT3 in HepG2 cells decreases MGAT activity by only ~35%, suggesting additional enzymes contribute to the activity

• Subcellular fractionation: Isolate crude mitochondrial membrane fractions, as MGAT2 and MGAT3 are enriched in mitochondrial-associated ER membranes rather than typical microsomes

• Substrate specificity analysis: Exploit differences in substrate preferences - MOGAT2 has higher activity with unsaturated fatty acids (C18:3 > C18:2 > C18:1 > C18:0)

• Genetic approaches: Complement inhibitor studies with siRNA knockdown or CRISPR knockout of specific acyltransferases to isolate individual contributions

• Combined approaches: For comprehensive analysis, employ both selective inhibitors and genetic approaches simultaneously to accurately assess MOGAT2's specific contribution to cellular lipid metabolism

What is the role of MOGAT2 in cancer progression and how can antibodies help investigate this?

Research reveals complex and context-dependent roles for MOGAT2 in cancer:

This research highlights the importance of tissue-specific context when investigating MOGAT2's role in cancer biology, with antibodies playing a crucial role in these investigations.

How does MOGAT2 ablation affect gut microbiota and intestinal health?

MOGAT2 knockout studies reveal significant effects on gut microbiota and intestinal health:

• Microbiota alterations:

  • Loss of Mogat2 leads to increased Verrucomicrobia at the phylum level

  • Heightened presence of Akkermansia muciniphila at the genus level, which constitutes 1-5% of normal gut microbiota

• Functional consequences:

  • Fecal microbiota transplantation (FMT) from Mogat2-/- mice to pseudo-germ-free mice promotes intestinal adenoma progression in Apc Min/+ mice

  • Significantly higher rates of Ki-67-positive cells (indicating increased proliferation) in intestinal tissues after FMT from Mogat2-/- mice

  • Reduced number of goblet cells per crypt in mice receiving FMT from Mogat2-/- mice, suggesting compromised intestinal barrier function

• Research methodologies:

  • Utilize whole-mount carmine alum staining to assess mammary gland and intestinal morphology in Mogat2-/- mice

  • Apply RT-PCR for gene expression analysis in various tissues (mammary tumors, stomach, small intestine, colorectal tissues)

  • Implement 16S rRNA sequencing to characterize microbial community changes

  • Perform histological analyses to evaluate tissue architecture and cellular composition

These findings suggest MOGAT2's effects on cancer progression may be partially mediated through microbiome alterations, highlighting the complex interplay between lipid metabolism, gut microbiota, and intestinal health.

How can MOGAT2 inhibitors be evaluated for therapeutic potential in metabolic disorders?

MOGAT2 inhibitors show promise for treating obesity and related metabolic disorders:

• Therapeutic rationale:

  • Loss of Mogat2 prevents high-fat diet-induced obesity in mice

  • MOGAT2 plays a central role in dietary fat absorption in the small intestine

  • Inhibition of MOGAT2 represents a potential strategy to reduce fat absorption and storage

• Screening methodologies:

  • Cell-based assays using stable isotope-labeled substrates and LC/MS for high sensitivity detection

  • In vitro enzyme assays using crude mitochondrial membrane fractions containing MOGAT2

  • Assays measuring incorporation of labeled fatty acids into diacylglycerol and triacylglycerol

• Efficacy evaluation:

  • Measure reduction in enzymatic activity and lipid synthesis in cellular models

  • Assess effects on lipid absorption and metabolism in animal models

  • Monitor key metabolic parameters (body weight, adiposity, glucose tolerance, insulin sensitivity)

• Selectivity assessment:

  • Test inhibitors against related enzymes (MGAT3, DGAT1, DGAT2) to ensure specificity

  • Evaluate potential off-target effects using broad screening panels

• Translation to human studies:

  • Investigate the translational potential in human tissues and cellular models

  • Design appropriate biomarkers for clinical studies (e.g., postprandial lipidemia)

The development of selective MOGAT2 inhibitors represents a promising approach for addressing obesity and related metabolic disorders, with cell-based assays providing crucial tools for screening and evaluation.

What methods can be used to investigate MOGAT2's role in different disease models?

Researchers can employ various approaches to study MOGAT2's function in disease models:

• Genetic models:

  • Mogat2 knockout mice (B6.129S4-Mogat2tm1Far/J) are commercially available for studying metabolic and cancer phenotypes

  • Tissue-specific conditional knockout models can isolate MOGAT2's role in specific organs

  • Combined models (e.g., Mogat2-/-PyMT) enable investigation of MOGAT2's impact on cancer development

• Molecular techniques:

  • RT-PCR to quantify MOGAT2 expression across tissues and disease states

  • Western blotting with validated antibodies to assess protein levels

  • Immunohistochemistry to evaluate tissue localization and expression patterns

• Functional assays:

  • ELISA-based detection of MOGAT2 protein in various biological samples (serum, plasma, cell culture supernatant, tissue lysates)

  • Enzyme activity assays measuring conversion of monoacylglycerol to diacylglycerol

  • Cell proliferation and colony formation assays for cancer models

• Translational approaches:

  • Analysis of MOGAT2 expression in patient-derived samples

  • Correlation of expression levels with clinical outcomes and disease progression

  • Microbiome analysis to assess effects of MOGAT2 modulation on gut microbial communities

These methodologies provide a comprehensive toolkit for investigating MOGAT2's multifaceted roles in various disease contexts, enabling researchers to identify potential therapeutic targets and biomarkers.

What are the considerations for using MOGAT2 as a prognostic marker in cancer research?

Using MOGAT2 as a prognostic marker requires careful consideration of several factors:

• Cancer-specific expression patterns:

  • MOGAT2 exhibits opposite prognostic significance in different cancers:

    • Negative prognostic factor in triple-negative breast cancer (higher expression = worse outcomes)

    • Positive prognostic factor in colorectal cancer (higher expression = better outcomes)

• Assessment methodology:

  • Immunohistochemical scoring systems (e.g., 13-point scale combining percentage of positive cells and staining intensity)

  • Western blot quantification normalized to appropriate housekeeping proteins

  • mRNA expression analysis through RT-PCR or RNA sequencing

  • Standardization of techniques is critical for consistent results across studies

• Integration with clinical parameters:

  • Correlation with tumor stage, grade, and molecular subtypes

  • Multivariate analysis to determine independent prognostic value

  • Combination with other established biomarkers for improved prognostic accuracy

• Validation requirements:

  • Multi-cohort validation with sufficient sample sizes

  • Prospective studies to confirm retrospective findings

  • Consistent antibody selection and standardized staining protocols

• Biological context:

  • Understanding of MOGAT2's context-dependent functions in different tissue types

  • Consideration of potential confounding factors (metabolic status, dietary patterns)

  • Evaluation of interplay with related metabolic pathways

The divergent prognostic significance of MOGAT2 in different cancer types underscores the importance of tissue-specific context and robust validation in biomarker development.

What are the potential limitations and troubleshooting approaches for MOGAT2 antibody applications?

When working with MOGAT2 antibodies, researchers should be aware of potential limitations and implement appropriate troubleshooting strategies:

• Specificity challenges:

  • MOGAT2 belongs to a family of related acyltransferases with structural similarities

  • Validate specificity using knockout/knockdown controls or peptide competition assays

  • Consider cross-reactivity with DGAT2 family members when interpreting results

• Detection sensitivity:

  • MOGAT2 may be expressed at low levels in some tissues

  • Optimize protein extraction methods for membrane-associated proteins

  • Consider signal amplification techniques for low-abundance targets

  • Use positive control samples (small intestine tissue) where MOGAT2 is known to be expressed

• Tissue-specific considerations:

  • Optimize fixation protocols for IHC applications (overfixation can mask epitopes)

  • For intestinal samples, avoid regions with high endogenous peroxidase activity

  • Consider specialized extraction buffers for lipid-rich tissues

• Western blot troubleshooting:

  • Observed molecular weight (36-38 kDa) may vary slightly from calculated weight (38 kDa)

  • Multiple bands may represent isoforms or post-translational modifications

  • Optimize membrane transfer conditions for hydrophobic proteins

• Reproducibility concerns:

  • Batch-to-batch variability may occur, especially with polyclonal antibodies

  • Include consistent positive controls across experiments

  • Document lot numbers and validate each new antibody lot

These technical considerations help ensure reliable results when working with MOGAT2 antibodies across various experimental applications.

What are the recommended controls for MOGAT2 antibody experiments?

Proper controls are essential for accurate interpretation of MOGAT2 antibody experiments:

• Positive tissue controls:

  • Small intestine tissue (mouse, rat, human) - primary site of MOGAT2 expression

  • HEK-293 cells - validated for Western blot and immunoprecipitation

• Negative controls:

  • Tissues with minimal MOGAT2 expression

  • Mogat2 knockout mouse tissues when available

  • Primary antibody omission controls for IHC/IF

• Expression controls:

  • Cells with confirmed overexpression of MOGAT2 (e.g., transfected HEK-293 cells)

  • siRNA or CRISPR knockout cells for antibody validation

  • Recombinant MOGAT2 protein standards for quantitative applications

• Technical controls:

  • Loading controls for Western blots (β-actin, GAPDH)

  • Isotype control antibodies for immunostaining applications

  • Secondary antibody-only controls to assess non-specific binding

• Cross-reactivity controls:

  • Related proteins (MOGAT1, MOGAT3, DGAT1, DGAT2) to assess specificity

  • Species cross-reactivity validation when using antibodies across different organisms

Including appropriate controls ensures robust and reliable interpretation of experimental results when working with MOGAT2 antibodies.