Magi2 Antibody

What is MAGI2 and where is it typically expressed?

MAGI2 (Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 2) is a scaffolding protein belonging to the MAGUK family. It is comprised of 6 PDZ domains, 2 WW domains, and 1 GUK domain, with a molecular weight of approximately 140-170 kDa . MAGI2 is primarily expressed in:

• Kidney podocytes, where it localizes to the filtration slit and colocalizes with Nephrin

• Brain tissue, particularly in neuronal post-synaptic membrane complexes

• Various other tissues where it mediates transport and signal transduction of G protein-coupled receptors (GPCRs)

MAGI2 serves as a molecular scaffold for multiple proteins, including alpha-actinin, dendrin, SMAD3, and beta-catenin, facilitating the signaling of both growth factor and neurotransmitter receptors such as ActRIIA, NMDA, and beta-1-adrenergic receptors .

What are the typical applications for MAGI2 antibodies in research?

Based on validated protocols, MAGI2 antibodies are commonly used in:

Western blot analysis typically reveals a specific band for MAGI2 at approximately 140-170 kDa . For immunofluorescence, MAGI2 antibodies show specific staining in the cytoplasm of U-87 MG human glioblastoma/astrocytoma cells .

What are the key species reactivity considerations when selecting MAGI2 antibodies?

Most commercial MAGI2 antibodies demonstrate reactivity across human, mouse, and rat samples . This cross-reactivity is particularly valuable for comparative studies, as MAGI2 is highly conserved across these species. When selecting an antibody:

• Verify the specific epitope region, as this may affect cross-reactivity

• Some antibodies are raised against fusion proteins (e.g., MAGI2 fusion protein Ag18279)

• Others target specific peptide sequences (e.g., human MAGI2 amino acids 221-270)

• For zebrafish studies, confirm reactivity as shown in some research applications

How should MAGI2 antibodies be stored and handled for optimal performance?

For optimal performance and longevity of MAGI2 antibodies:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Most formulations contain glycerol (typically 50%) and PBS with 0.02% sodium azide

• After reconstitution, antibodies are typically stable for:

  • 1 month at 2-8°C under sterile conditions

  • 6 months at -20 to -70°C under sterile conditions

• Some preparations contain BSA (0.1%) for added stability

• Ensure proper handling during experiments to maintain activity

What are the recommended protocols for Western blot detection of MAGI2?

For optimal Western blot detection of MAGI2:

• Sample preparation:

  • Use brain tissue (particularly cortex) or kidney tissue for highest expression

  • Lyse tissues in buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 2 mM MgCl₂, 1% Triton, and protease inhibitor cocktail

• Gel electrophoresis and transfer:

  • Use reducing conditions

  • PVDF membrane is recommended for optimal protein binding

• Antibody incubation:

  • Primary antibody dilution: 1:5000-1:50000 (antibody-dependent)

  • For R&D Systems AF7117: use 1 μg/mL concentration

  • Secondary antibody: HRP-conjugated anti-species IgG

• Detection:

  • Expect a specific band at approximately 140-170 kDa

  • Use Immunoblot Buffer Group 1 for optimal results with some antibodies

What are the optimal approaches for immunofluorescence detection of MAGI2?

For immunofluorescence applications:

• Sample preparation:

  • Cell lines: U-87 MG human glioblastoma/astrocytoma cells show good expression

  • Tissue sections: Brain and kidney sections are recommended

• Fixation method:

  • Immersion fixation is commonly used for both cells and tissues

  • For paraffin-embedded sections, standard deparaffinization protocol is required

• Antibody incubation:

  • Dilution range: 1:50-1:500 for most antibodies

  • For R&D Systems AF7117: use 10 μg/mL for 3 hours at room temperature

  • Secondary antibody: Fluorophore-conjugated anti-species IgG (e.g., NorthernLights™ 557-conjugated Anti-Goat IgG)

• Counterstaining:

  • DAPI for nuclear visualization

  • In kidney sections, co-staining with Nephrin antibodies helps visualize colocalization at the filtration slit

How can MAGI2 antibodies be used to study protein-protein interactions?

MAGI2 serves as a scaffold for multiple proteins, making protein interaction studies valuable. Methodological approaches include:

• GST-Pull-Down Assay:

  • Express Flag-tagged full-length MAGI2 in HEK293T cells

  • Lyse cells in buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 2 mM MgCl₂, 1% Triton, and protease inhibitor cocktail

  • Incubate lysates with GST-tagged protein fragments (e.g., GST-Nephrin) preloaded on GSH-Sepharose beads

  • After washing, detect captured MAGI2 by western blot using anti-Flag antibody (1:3000)

• Co-immunoprecipitation:

  • MAGI2 antibodies can be used to pull down protein complexes

  • Verify interactions with antibodies against suspected binding partners

  • For crystallography studies of complexes (e.g., MAGI2 WW1–2/Dendrin complex), specific protocols involving co-purification have been developed

How can MAGI2 antibodies be applied in studies of kidney podocyte biology?

MAGI2 is crucial in podocyte biology, with important implications for kidney research:

• Immunohistochemistry of kidney sections:

  • MAGI2 localizes to the filtration slit of podocytes and colocalizes with Nephrin

  • Super-resolution microscopy (3D-SIM) provides detailed visualization of this colocalization

  • For optimal results, use confocal-LSM imaging of anti-MAGI2 stained kidney sections

• Quantitative analysis in disease models:

  • Deep-Learning networks (U-Net) have been applied to segment and analyze MAGI2 expression in glomerular disease models

  • Monitor MAGI2 downregulation in podocyte dedifferentiation models

  • Progression of MAGI2 loss can be tracked using both protein (immunostaining) and mRNA (RT-qPCR) levels

What are the considerations when studying MAGI2 phase separation properties?

Recent research has shown that MAGI2 undergoes liquid-liquid phase separation, which can be studied using specialized approaches:

• Protein purification:

  • For studies of phase separation properties, highly purified proteins are required

  • Tag cleavage by specific proteases (e.g., human rhinovirus 3C protease) helps eliminate tag effects

• Visualization techniques:

  • Fluorescently tagged MAGI2 constructs

  • Differential interference contrast microscopy

  • Fluorescence recovery after photobleaching (FRAP) to analyze dynamics

• Modulation of phase separation:

  • Study effects of binding partners on MAGI2 phase separation

  • Analyze how post-translational modifications affect this property

  • Temperature and salt concentration dependencies

How can MAGI2 antibodies be used in inflammatory bowel disease (IBD) research?

MAGI2 has been implicated in IBD pathogenesis, and antibodies can be valuable tools:

• Genetic variation analysis:

  • MAGI2 variants have been associated with IBD, particularly in intron 6 (rs2160322)

  • Antibodies can help correlate genotype with protein expression levels

• Tissue expression studies:

  • Compare MAGI2 expression in control versus IBD patient samples

  • Analyze colocalization with other tight junction proteins

  • Correlate expression with disease severity and progression

• Intermediate phenotype analysis:

  • MAGI2 variants influence antibody production to microbial antigens

  • Antibodies can help establish mechanistic links between genetic variations and disease phenotypes

What controls should be included when using MAGI2 antibodies in disease models?

Proper controls are essential for meaningful results in disease-related research:

• Tissue-specific controls:

  • Brain cortex and kidney samples as positive controls

  • Non-expressing tissues as negative controls

  • For kidney studies, include samples from various nephron segments

• Antibody validation controls:

  • Blocking peptides specific to the antibody epitope

  • MAGI2 knockout or knockdown samples when available

  • Isotype controls (e.g., Rabbit IgG) at matching concentrations

• Disease model-specific controls:

  • Time-course samples for progression studies

  • For podocyte dedifferentiation models, compare with other podocyte markers (Nephrin, Podocin, Synaptopodin, Alpha-actinin-4)

  • Include both protein and mRNA level analyses for comprehensive assessment

What are common challenges when using MAGI2 antibodies and how can they be addressed?

How can researchers validate the specificity of their MAGI2 antibody?

Comprehensive validation strategies include:

• Multiple application testing:

  • Verify consistent results across WB, IF/ICC, and IHC applications

  • Compare observed molecular weight (140-170 kDa) with expected weight (calculated: 159 kDa)

• Cross-platform verification:

  • Compare protein expression with mRNA expression data

  • Verify with single-cell RNA sequencing data showing MAGI2 clustering in relevant cell types

• Epitope mapping and competition assays:

  • Use immunogenic peptides to block antibody binding

  • Test antibodies targeting different epitopes of MAGI2

  • Compare commercial antibodies from different sources

• Genetic manipulation:

  • Use MAGI2 knockdown or knockout samples as negative controls

  • Test overexpression systems for increased signal