MEGF10 Antibody

What types of MEGF10 antibodies are available for research applications?

Researchers can choose from several types of MEGF10 antibodies depending on their experimental needs. Rabbit polyclonal antibodies are commonly available for detecting MEGF10, with validated applications in Western Blotting and Immunofluorescence . Domain-specific antibodies targeting the extracellular region, like ANR-205 which recognizes amino acid residues 178-190 of mouse MEGF10, provide options for experiments requiring detection of the protein's external portion . These antibodies are typically unconjugated, allowing researchers flexibility in secondary detection methods.

The most widely validated applications include:

• Western Blotting (WB): Recommended dilutions typically range from 1:300-5000

• Immunofluorescence (IF): Effective for tissue sections and cultured cells

• Flow cytometry: Particularly for antibodies targeting extracellular domains

How should researchers evaluate antibody specificity for MEGF10 detection?

Rigorous validation of antibody specificity is critical for reliable MEGF10 research. Recommended validation approaches include:

• Blocking peptide experiments: Pre-incubation with specific blocking peptides (e.g., BLP-NR205 for ANR-205 antibody) should eliminate signal if the antibody is specific .

• Genetic controls: Tissues from MEGF10 knockout models (Megf10−/−) serve as definitive negative controls.

• Positive control samples: Several cell lines have been validated for MEGF10 expression, including:

  • Human U-87 MG glioblastoma

  • Human MCF-7 breast adenocarcinoma

  • Human 1311N1 astrocytoma

  • THP-1 monocytic leukemia cells

• Cross-validation: Comparing results using multiple antibodies targeting different MEGF10 epitopes increases confidence in findings.

What methodologies are most effective for studying MEGF10's role in phagocytosis?

MEGF10 functions as a receptor for C1q and mediates clearance of apoptotic cells, particularly in the developing brain . To investigate this function:

• Phagocytosis assays: Transfect cells with MEGF10 expression vectors and quantify their ability to engulf labeled apoptotic cells. HEK-293T cells transfected with mMegf10-RFP or hMegf10-GFP have been successfully used for this purpose .

• Purified domain studies: Express and purify the extracellular domain of MEGF10 (ex-hMegf10) and use in binding assays with potential ligands like C1q. This approach has revealed direct high-affinity binding between MEGF10 and C1q .

• Apoptotic cell clearance measurement: Compare TUNEL-positive cell counts in tissues from Megf10+/+, Megf10+/−, and Megf10−/− animals to assess in vivo phagocytic function .

• Mutation analysis: Compare wild-type MEGF10 with disease-associated mutants (e.g., C326R and C774R EMARDD mutations) in phagocytic assays to understand structure-function relationships .

How can MEGF10 antibodies be utilized to investigate its role in myoblast development?

MEGF10 plays a significant role in muscle development, with mutations causing EMARDD (early myopathy, areflexia, respiratory distress and dysphagia) . Methodological approaches include:

• Myoblast fusion assays: Monitor MEGF10 localization during fusion events using immunofluorescence. Studies have shown that MEGF10 overexpression inhibits fusion in H-2k-tsA58 myoblasts .

• Domain function analysis: Add purified extracellular domains of MEGF10 to myoblast cultures and assess effects on adhesion and fusion. The full extracellular domain (ECD) with the EMI domain reduces myoblast fusion more effectively than EGF domains alone .

• Satellite cell quantification: Use MEGF10 antibodies alongside PAX7 to quantify satellite cell populations in normal versus EMARDD muscle samples.

• Hypertrophic response models: Analyze MEGF10 expression during muscle overload to understand its role in adaptation responses .

What approaches should be used to study MEGF10-C1q interactions in neurological contexts?

MEGF10 functions as a receptor for C1q in the clearance of apoptotic cells in the developing brain . To investigate this interaction:

• Binding assays: Expressed and purified extracellular domains of human MEGF10 (ex-hMegf10) have been used to demonstrate direct binding to C1q. This can be accomplished by:

  • Immobilizing C1q on membranes and detecting binding with purified MEGF10 domains

  • Using anti-Flag detection for tagged MEGF10 constructs

  • Employing chemiluminescent detection systems

• Mutation analysis: Compare C1q binding between wild-type MEGF10 and EMARDD-associated mutants (C326R and C774R). Cells expressing MEGF10 with these mutations show impaired apoptotic cell clearance and reduced C1q binding .

• Flow cytometry quantification: Measure the percentage of C1q uptake in cells expressing different MEGF10 variants to quantify functional interactions .

• Co-localization studies: Use dual immunofluorescence with anti-MEGF10 and anti-C1q antibodies to examine spatial relationships in tissue sections.

What are the optimal techniques for investigating MEGF10 in different neural cell populations?

To effectively study MEGF10 expression and function across neural cell types:

• Cell-type specific co-localization: Combine anti-MEGF10 antibodies with markers for:

  • Neurons: MEGF10 immunoreactivity has been detected in neurons of the rat substantia nigra

  • Glia: Multiple glial cell lines express MEGF10, including U-87 MG glioblastoma and 1311N1 astrocytoma cells

• Subcellular localization: High-resolution imaging of MEGF10 demonstrates localization patterns including:

  • Cell membrane (primary location)

  • Intracellular vesicles

  • Golgi apparatus (particularly at higher expression levels)

• Flow cytometry: For cell surface detection of MEGF10, use antibodies targeting extracellular epitopes in live intact cells (as demonstrated with THP-1 cells) .

• Tissue section analysis: Perfusion-fixed frozen brain sections can be effectively stained with anti-MEGF10 antibodies at dilutions of approximately 1:300, allowing visualization of neuronal MEGF10 expression .

What are the optimal conditions for Western blotting with MEGF10 antibodies?

For successful Western blot detection of MEGF10, researchers should consider:

• Sample preparation:

  • For tissue samples: Mouse or rat brain membranes work effectively

  • For cell lines: U-87 MG, MCF-7, 1311N1, and THP-1 cells all express detectable MEGF10

• Antibody dilutions:

  • ANR-205: Effective at 1:400 dilution for Western blotting

  • bs-24335R: Recommended range of 1:300-5000

• Detection systems:

  • For rabbit-derived antibodies: Anti-rabbit HRP-conjugated secondary antibodies

  • Visualization: Chemiluminescent detection systems

• Controls:

  • Blocking peptide controls should be run in parallel to confirm specificity

  • Include positive control lysates from validated MEGF10-expressing cells

How should researchers interpret different patterns of MEGF10 reactivity across species?

When working with MEGF10 antibodies across different species:

• Cross-reactivity profiles:

  • Anti-MEGF10 antibody bs-24335R reacts with human MEGF10 and is predicted to recognize mouse, rat, cow, sheep, pig, horse, chicken, and rabbit variants

  • The source peptide used for antibody development affects species specificity (e.g., ANR-205 targets mouse MEGF10 amino acids 178-190)

• Conservation analysis:

  • The EMI domain and EGF repeats show different degrees of conservation across species

  • Functional studies indicate the EMI domain plays an important role in adhesion and fusion behaviors

• Validation across species:

  • Western blot analysis has confirmed detection of MEGF10 in both mouse and rat brain membranes using the same antibody

  • Antibodies raised against human MEGF10 may have variable reactivity with orthologs

How can researchers effectively study the role of MEGF10 in pathological conditions?

MEGF10 mutations cause EMARDD, making it relevant for pathology research . Methodological approaches include:

• Mutation modeling: Compare wild-type MEGF10 with disease-associated mutations:

  • C326R and C774R mutations impair apoptotic cell clearance and C1q binding

  • Expression systems using HEK-293T cells can effectively model these functional differences

• Satellite cell analysis: EMARDD is associated with reduced numbers of PAX7-positive satellite cells . Researchers can:

  • Quantify satellite cell populations in muscle biopsies

  • Correlate MEGF10 expression with satellite cell function

• Overload models: Muscle overload studies in Megf10 knockout mice reveal the protein's role in hypertrophic responses, providing insight into disease mechanisms .

• Domain-specific functions: Purified extracellular domains demonstrate that the EMI domain is particularly important for adhesion behaviors, while its absence affects fusion patterns .

What methodologies are recommended for studying MEGF10 protein-protein interactions?

To investigate MEGF10's interactome:

• Direct binding assays:

  • Express and purify extracellular domains using 6×His–3×Flag tags

  • Purify using nickel columns (elute with 0.25 M imidazole)

  • Confirm purification by Western blotting with anti-MEGF10 or anti-Flag antibodies

  • Use purified protein in binding assays with potential partners like C1q

• Membrane interaction studies:

  • Membrane lipid strips spotted with defined lipids (100 pmol)

  • Block with 5% BSA in TBST

  • Incubate with purified MEGF10 domains (15 μg)

  • Detect binding with anti-Flag antibodies

• Functional interaction analysis:

  • MEGF10 cooperates with ABCA1 during engulfment

  • MEGF10 destabilizes oligomeric assemblies of the ABCA1 transporter

  • These interactions can be studied through co-immunoprecipitation and functional assays

What approaches are recommended for studying the differential effects of MEGF10 domains?

Research has revealed distinct functions for MEGF10 domains, particularly the EMI domain versus EGF repeats . Methodological approaches include:

• Domain-specific constructs:

  • Full extracellular domain (ECD): Contains EMI domain plus EGF repeats

  • EGF-only constructs: Lack the N-terminal EMI domain

  • These constructs show different effects on myoblast adhesion and fusion

• Quantitative adhesion assays:

  • ECD promotes adhesion to non-adhesive surfaces

  • This effect differs from EGF-only constructs, highlighting the EMI domain's importance

• Fusion kinetics:

  • The ECD more effectively reduces myoblast fusion by day 7 of differentiation

  • Time-course experiments reveal domain-specific effects on the rate of fusion

How can researchers utilize MEGF10 antibodies to study developmental processes?

To investigate MEGF10's role in development:

• Developmental time-course studies:

  • MEGF10 plays a role in clearance of apoptotic cells in the developing brain

  • Stage-specific analysis of expression patterns can reveal temporal regulation

• Cell fate determination:

  • MEGF10 inhibits myoblast fusion

  • This function may prevent premature differentiation of satellite cells

  • Antibody staining at different developmental stages can track this regulatory function

• Genetic model analysis:

  • Compare development in Megf10+/+, Megf10+/−, and Megf10−/− animals

  • Assess satellite cell counts and muscle development

  • Correlate with functional outcomes and adaptation responses