MAGI2 Antibody, FITC conjugated

What is MAGI2 and why is it important in research?

MAGI2 (also known as Atrophin-1-interacting protein 1, S-SCAM, or ARIP1) is a scaffold protein belonging to the membrane-associated guanylate kinase (MAGUK) family. It contains multiple protein-protein interaction domains including WW and PDZ domains, facilitating complex formation with various binding partners. MAGI2 is specifically expressed in brain tissue and plays a critical role in the formation of slit diaphragms between podocytes in the kidney . Recent research has revealed MAGI2's ability to undergo liquid-liquid phase separation, which appears fundamental to the formation of electron-dense compartments at slit diaphragms . This property makes MAGI2 particularly interesting for studying compartmentalized signaling and the biophysical properties of membrane-associated protein complexes.

What are the known binding partners and functions of MAGI2?

MAGI2 functions as a scaffold molecule that assembles multi-protein complexes, particularly at synaptic junctions and slit diaphragms. Research has identified several key binding partners:

• Dendrin - Interacts with MAGI2 through its PY motifs binding to MAGI2's WW domains

• CD2AP - Forms part of the MAGI2-Dendrin-CD2AP complex at slit diaphragms

• Nephrin - Recruited to condensates formed by the MAGI2-Dendrin-CD2AP complex

This network of interactions facilitates the formation of electron-dense protein-rich compartments that are essential for maintaining slit diaphragm integrity and podocyte signal transduction. Disruptions in these interactions have been linked to nephrotic syndrome and other glomerular diseases .

What is the difference between standard MAGI2 antibodies and FITC-conjugated versions?

Standard MAGI2 antibodies require secondary detection methods (such as secondary antibodies conjugated to fluorophores, HRP, or other detection molecules), whereas FITC-conjugated MAGI2 antibodies have fluorescein isothiocyanate directly attached to the antibody molecule. This direct labeling offers several advantages:

• Direct visualization without secondary antibody steps

• Reduction in background signal and non-specific binding associated with secondary antibodies

• Compatibility with multi-labeling experiments using antibodies from the same host species

• Simplified workflow and reduced experimental time

The FITC-conjugated anti-MAGI2 antibody (e.g., ABIN7159358) is particularly useful for immunofluorescence applications, targeting amino acids 1308-1455 of human MAGI2 with high specificity .

What are the typical applications for MAGI2 antibodies in research?

MAGI2 antibodies are particularly valuable for studying kidney podocyte biology, neuronal synapses, and diseases associated with mutations in the MAGI2 gene .

How can FITC-conjugated MAGI2 antibodies help investigate the liquid-liquid phase separation properties of MAGI2?

Recent research has revealed that MAGI2 undergoes liquid-liquid phase separation (LLPS) both in vitro and in living cells, forming condensates that are critical for slit diaphragm assembly in kidney podocytes . FITC-conjugated MAGI2 antibodies provide a powerful tool for investigating this phenomenon through several methodological approaches:

• Live imaging of phase separation dynamics: FITC-conjugated antibodies can be used to visualize the formation, fusion, and dissolution of MAGI2 condensates in real-time in live cells, providing insights into the kinetics of phase separation.

• Colocalization studies: By combining FITC-conjugated MAGI2 antibodies with differently labeled markers for binding partners (such as Dendrin and CD2AP), researchers can examine how these proteins co-partition into condensates and study the multivalent interactions that drive condensate formation.

• FRAP (Fluorescence Recovery After Photobleaching) analysis: The fluorescent properties of FITC-conjugated antibodies enable FRAP experiments to measure the mobility of MAGI2 within condensates, offering insights into the material properties of these biomolecular assemblies.

• Effect of disease mutations: FITC-conjugated antibodies can help visualize how nephrotic syndrome-associated mutations in MAGI2 affect condensate formation, potentially revealing mechanisms of disease pathogenesis .

This approach has revealed that paralog-specific "RQPPxxxDY" repetitive motifs in MAGI2 are essential for its phase separation properties, distinguishing it from other family members like MAGI1 .

How do the binding properties of MAGI2-Dendrin-CD2AP complexes influence experimental design using FITC-conjugated MAGI2 antibodies?

The multivalent interactions between MAGI2, Dendrin, and CD2AP create a complex binding network that drives the formation of condensates at slit diaphragms . This has several implications for experiments using FITC-conjugated MAGI2 antibodies:

• Epitope accessibility: The targeted epitope (aa 1308-1455) in FITC-conjugated anti-MAGI2 antibodies must remain accessible when MAGI2 is engaged in complexes with Dendrin and CD2AP. Researchers should verify that antibody binding doesn't disrupt or is not hindered by these protein-protein interactions.

• Concentration thresholds: Research has shown that the concentration threshold for MAGI2 condensate formation decreases when Dendrin (10 μM) and CD2AP (5 μM) are present . Experiments should account for these concentration-dependent effects, especially when studying dilute samples.

• Buffer conditions: Phase separation is highly sensitive to buffer conditions (salt concentration, pH, temperature). Optimization of imaging conditions with FITC-conjugated antibodies should consider these parameters to accurately capture physiologically relevant condensates.

• Temporal dynamics: The MAGI2-Dendrin-CD2AP complex forms dynamic condensates. Time-lapse imaging using FITC-conjugated antibodies should be designed to capture the assembly and disassembly kinetics, which may occur on different timescales.

Understanding these complexities helps researchers design more rigorous experiments and accurately interpret results when using FITC-conjugated MAGI2 antibodies to study these multicomponent biomolecular assemblies.

What are the considerations for using FITC-conjugated MAGI2 antibodies in multi-color imaging experiments?

Multi-color imaging using FITC-conjugated MAGI2 antibodies requires careful experimental design:

• Spectral properties: FITC has excitation/emission maxima at approximately 495/519 nm. When designing multi-color experiments, select fluorophores with minimal spectral overlap, such as:

  • DAPI (Ex/Em: 358/461 nm) for nuclei

  • TRITC (Ex/Em: 557/576 nm) or Cy3 (Ex/Em: 550/570 nm) for additional proteins

  • Cy5 (Ex/Em: 650/670 nm) for third markers

• Bleed-through prevention: Implement appropriate controls and sequential scanning techniques to minimize bleed-through between channels, particularly important when studying the colocalization of MAGI2 with its binding partners.

• Fixation considerations: FITC fluorescence can be sensitive to certain fixation methods. Paraformaldehyde (4%) is generally compatible, while methanol fixation may reduce signal intensity. Test fixation protocols to optimize signal preservation.

• Antibody combinations: When studying MAGI2 interactions with Dendrin and CD2AP in the same sample, select antibodies raised in different host species to enable simultaneous detection without cross-reactivity. For example:

  • FITC-conjugated rabbit anti-MAGI2

  • Mouse anti-Dendrin with Cy3-conjugated secondary

  • Goat anti-CD2AP with Cy5-conjugated secondary

• Photobleaching management: FITC is moderately susceptible to photobleaching. Consider using anti-fade mounting media and acquiring FITC images first when using sequential imaging approaches.

These considerations will help ensure accurate visualization and quantification of MAGI2 and its binding partners in complex cellular contexts.

How do MAGI2 mutations associated with nephrotic syndrome affect antibody binding and experimental outcomes?

Nephrotic syndrome-associated mutations in MAGI2 can interfere with the protein's ability to form phase-separated condensates and properly recruit binding partners like Nephrin . This has important implications for antibody-based detection:

• Epitope alterations: Mutations may directly affect antibody epitopes, potentially reducing binding affinity or completely preventing recognition. Researchers should verify whether the FITC-conjugated antibody's target region (aa 1308-1455) includes or is structurally affected by known disease mutations.

• Conformational changes: Even mutations distant from the antibody epitope may cause conformational changes that alter epitope accessibility. This can result in differential staining efficiency between wild-type and mutant MAGI2.

• Localization differences: Disease-associated mutations may alter MAGI2's subcellular localization. When using FITC-conjugated antibodies to compare wild-type and mutant MAGI2 distribution, researchers should account for these potential differences when interpreting results.

• Complex formation: Mutations that disrupt the MAGI2-Dendrin-CD2AP complex may affect co-immunostaining patterns. Controls comparing staining of individual proteins versus the complex are essential for accurate interpretation.

• Quantification challenges: When quantifying fluorescence intensity to compare wild-type and mutant MAGI2 levels, researchers must determine whether differences reflect actual protein abundance changes or alterations in antibody accessibility due to structural changes.

Understanding these mutation-specific effects is crucial for correctly interpreting experimental results, particularly in disease model systems or patient-derived samples.

What are the optimal sample preparation protocols for FITC-conjugated MAGI2 antibody applications?

Optimal sample preparation is crucial for successful experiments with FITC-conjugated MAGI2 antibodies:

For cellular immunofluorescence (IF/ICC):

• Fixation: Use 4% paraformaldehyde in PBS for 15-20 minutes at room temperature to preserve cellular structures while maintaining epitope accessibility.

• Permeabilization: Apply 0.1-0.3% Triton X-100 in PBS for 5-10 minutes to allow antibody access to intracellular MAGI2.

• Blocking: Incubate with 5% normal serum (from species not related to primary antibody source) and 1% BSA in PBS for 1 hour to reduce non-specific binding.

• Antibody dilution: Dilute FITC-conjugated MAGI2 antibody in blocking solution. While optimal dilutions must be determined empirically, a starting range of 1:50-1:200 is recommended .

• Incubation conditions: For best results, incubate overnight at 4°C in a humidified chamber protected from light to preserve FITC fluorescence.

For tissue sections:

• Section preparation: Use 5-8 μm sections mounted on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) to maximize epitope accessibility.

• Autofluorescence reduction: Treat with 0.1% sodium borohydride or commercial autofluorescence quenchers to minimize background, particularly important with kidney tissue.

For phase separation studies:

• Buffer composition: Use physiological buffers (e.g., 150 mM NaCl, 20 mM HEPES, pH 7.4) to maintain native MAGI2 phase separation properties .

• Protein concentration: Ensure MAGI2 concentration exceeds the phase separation threshold (≥25 μM for MAGI2 alone, ≥10 μM with Dendrin, or ≥5 μM with Dendrin and CD2AP) .

Careful optimization of these parameters will yield the most reliable and interpretable results with FITC-conjugated MAGI2 antibodies.

How should researchers validate the specificity of FITC-conjugated MAGI2 antibodies?

Rigorous validation of FITC-conjugated MAGI2 antibody specificity is essential for reliable experimental results:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (MAGI2 aa 1308-1455) before application to samples. Specific staining should be significantly reduced or eliminated.

• Knockout/knockdown controls: Compare staining patterns between wild-type samples and those with MAGI2 knockout (CRISPR/Cas9) or knockdown (siRNA/shRNA). Specific signals should be absent or significantly reduced in knockout/knockdown samples.

• Recombinant protein controls: Test antibody binding to purified recombinant MAGI2 protein versus irrelevant proteins by dot blot or ELISA to confirm specificity.

• Cross-reactivity assessment: Test the antibody on tissues from different species to confirm reactivity matches the predicted pattern (e.g., human, mouse, rat) .

• Orthogonal detection methods: Verify MAGI2 localization using alternative detection methods:

  • Compare FITC-conjugated antibody staining with unconjugated MAGI2 antibodies detected with secondary antibodies

  • Correlate with GFP-tagged MAGI2 expression patterns

  • Confirm with in situ hybridization for MAGI2 mRNA

• Western blot correlation: Confirm that FITC-conjugated antibody recognizes a single band of the expected size (159 kDa) in Western blot assays from the same samples used for immunofluorescence.

• Analysis of known expression patterns: Verify that staining patterns match known MAGI2 expression (e.g., specific expression in brain and kidney podocytes) .

Documented validation using multiple approaches provides confidence in experimental results and should be included in research publications.

What are the optimal imaging parameters for detecting FITC-conjugated MAGI2 antibodies in confocal microscopy?

Optimizing imaging parameters is critical for accurate visualization and quantification of FITC-conjugated MAGI2 antibodies:

• Excitation/emission settings:

  • Excitation: 488-495 nm laser line

  • Emission collection: 505-530 nm bandpass filter

  • Dichroic mirror: 500-505 nm

• Microscope configuration:

  • Pinhole: 1 Airy unit for optimal confocality

  • Objective: High NA (≥1.3) objectives for maximum resolution

  • Scan speed: Slower scanning (≤400 Hz) for improved signal-to-noise ratio

• Signal optimization:

  • Detector gain: Set to maximize signal while avoiding saturation

  • Laser power: Use minimum necessary power (typically 2-5%) to minimize photobleaching

  • Line/frame averaging: Apply 2-4× line averaging to improve signal-to-noise ratio

• Image acquisition strategy:

  • Z-stack parameters: 0.3-0.5 μm step size for optimal sampling

  • Pixel size: Set to 2-3× oversampling (typically ~0.1 μm/pixel with 63× objective)

  • Dynamic range: Acquire 12-16 bit images to capture full intensity range

• Phase separation-specific considerations:

  • Time-lapse parameters: For studying MAGI2 condensate dynamics, acquire frames at 5-30 second intervals

  • Temperature control: Maintain physiological temperature (37°C) during live imaging as phase separation is temperature-dependent

  • Environmental chamber: Control CO₂ (5%) and humidity to maintain physiological conditions

• Multi-channel acquisition for MAGI2 complex imaging:

  • Sequential scanning: Capture channels separately to prevent bleed-through

  • Channel order: Acquire FITC channel first to minimize photobleaching effects

  • Reference markers: Include nuclear counterstain (DAPI) for consistent image alignment and analysis

These optimized parameters will enable high-quality imaging of FITC-conjugated MAGI2 antibody staining, particularly important for visualizing the subtle dynamics of phase-separated MAGI2 condensates.

How can researchers quantitatively analyze MAGI2 phase separation using FITC-conjugated antibodies?

Quantitative analysis of MAGI2 phase separation using FITC-conjugated antibodies requires rigorous methodological approaches:

• Condensate detection and measurement:

  • Apply intensity thresholding to identify condensates (typically 2-3× above background)

  • Measure condensate parameters: number, size distribution, circularity, and mean/integrated intensity

  • Track temporal changes in these parameters for dynamic studies

• Partition coefficient calculation:

  • Measure the ratio of FITC intensity inside condensates versus the surrounding dilute phase

  • Higher partition coefficients indicate stronger enrichment of MAGI2 in condensates

  • Compare partition coefficients across experimental conditions (e.g., wild-type vs. mutant MAGI2)

• Colocalization analysis with binding partners:

  • Calculate Pearson's correlation coefficient and Manders' overlap coefficient between FITC-MAGI2 and labeled binding partners (Dendrin, CD2AP, Nephrin)

  • Apply intensity correlation analysis to quantify the spatial relationship between components

  • Use object-based colocalization to determine the percentage of MAGI2 condensates containing specific binding partners

• FRAP analysis for material properties:

  • Photobleach a region within FITC-labeled MAGI2 condensates

  • Measure fluorescence recovery over time to calculate:

    • Mobile fraction (percentage of MAGI2 that can exchange)

    • Half-time of recovery (indicative of molecular mobility within condensates)

  • Compare recovery parameters between different conditions or mutations

• Concentration dependence assessment:

  • Systematically vary protein concentrations to determine phase separation thresholds

  • Plot concentration-dependent changes in condensate formation

  • Compare these values to the published thresholds (25 μM for MAGI2 alone, 10 μM with Dendrin, 5 μM with Dendrin and CD2AP)

• Image analysis software recommendations:

  • ImageJ/Fiji with Comdet plugin for condensate detection

  • CellProfiler for automated high-throughput analysis

  • Imaris for 3D visualization and analysis

  • Custom MATLAB or Python scripts for specialized analyses

These quantitative approaches enable rigorous characterization of MAGI2 phase separation properties and how they are affected by disease mutations or experimental perturbations.

What are common issues encountered with FITC-conjugated MAGI2 antibodies and how can they be resolved?

How can researchers optimize FITC-conjugated MAGI2 antibody staining to visualize interactions with Dendrin and CD2AP?

To optimize visualization of MAGI2-Dendrin-CD2AP complexes in phase-separated condensates:

• Sequential immunostaining approach:

  • First apply FITC-conjugated MAGI2 antibody

  • Follow with unconjugated antibodies against Dendrin and CD2AP

  • Detect with spectrally distinct secondary antibodies (e.g., Cy3 and Cy5)

  • This approach minimizes potential steric hindrance between antibodies

• Antibody concentration balancing:

  • Titrate antibody concentrations to achieve comparable signal intensities across channels

  • Typical ratios: 1:100 FITC-MAGI2, 1:200 anti-Dendrin, 1:150 anti-CD2AP

  • Equal visual intensities facilitate more accurate colocalization analysis

• Sample preparation refinements:

  • Mild fixation (2% PFA for 10 minutes) better preserves condensate structure

  • Gentle permeabilization (0.1% Triton X-100 for 5 minutes) maintains phase-separated architecture

  • Extended blocking (2+ hours) with 5% BSA reduces non-specific binding

• Advanced visualization techniques:

  • Apply super-resolution microscopy (STED, STORM) to resolve fine structure within condensates

  • Implement Airyscan detection for improved resolution with reduced photobleaching

  • Consider lattice light-sheet microscopy for rapid 3D imaging of live condensate dynamics

• Controls for interaction specificity:

  • Include samples with individual proteins expressed separately

  • Compare wild-type MAGI2 with phase separation-deficient mutants

  • Use proximity ligation assay (PLA) to verify direct protein-protein interactions within condensates

These optimizations enable detailed visualization of the MAGI2-Dendrin-CD2AP complex and its role in forming phase-separated condensates at slit diaphragms.

How does the buffer composition affect FITC-conjugated MAGI2 antibody performance in phase separation studies?

Buffer composition significantly impacts MAGI2 phase separation properties and consequently affects FITC-conjugated antibody performance:

• Salt concentration effects:

  • Physiological salt (150 mM NaCl) supports proper MAGI2 phase separation

  • Higher salt concentrations (>200 mM) disrupt electrostatic interactions and can dissolve condensates

  • Lower salt (<100 mM) may promote non-specific aggregation rather than true phase separation

  • Recommendation: Standardize to 150 mM NaCl for consistency across experiments

• pH considerations:

  • FITC fluorescence is pH-sensitive, with optimal emission at pH 7.4-8.0

  • MAGI2 phase separation is also pH-dependent, with optimal formation at physiological pH (7.2-7.4)

  • pH below 6.5 can significantly reduce FITC signal intensity

  • Recommendation: Maintain strict pH control at 7.4 for both phase separation and optimal FITC visualization

• Crowding agent influences:

  • Molecular crowding enhances MAGI2 phase separation

  • PEG-3350 (5-10%) or Ficoll (10-15%) can lower the concentration threshold for condensate formation

  • Crowding agents may affect antibody diffusion and binding kinetics

  • Recommendation: If using crowding agents, maintain consistent concentrations across experiments

• Divalent cation effects:

  • Ca²⁺ and Mg²⁺ (1-2 mM) can stabilize MAGI2 condensates

  • EDTA or EGTA may disrupt condensate formation by chelating essential cations

  • Recommendation: Include 1 mM MgCl₂ and 1 mM CaCl₂ in buffers for stable condensates

• Reducing agent considerations:

  • DTT or β-mercaptoethanol may affect FITC fluorescence

  • Mild reducing conditions (0.5-1 mM DTT) help maintain protein stability without significantly impacting fluorescence

  • Recommendation: Include 0.5 mM DTT in buffers for optimal protein stability

• Optimal buffer composition for FITC-MAGI2 phase separation studies:

  • 20 mM HEPES pH 7.4

  • 150 mM NaCl

  • 1 mM MgCl₂

  • 1 mM CaCl₂

  • 0.5 mM DTT

  • 5% glycerol

  • 0.01% NP-40 (to reduce non-specific antibody binding)

This optimized buffer composition supports physiological MAGI2 phase separation while maintaining FITC fluorescence properties and antibody binding capacity.

What are the considerations for studying MAGI2-associated disease mutations using FITC-conjugated antibodies?

When investigating MAGI2 mutations associated with nephrotic syndrome and other diseases using FITC-conjugated antibodies, researchers should consider:

• Mutation-specific effects on antibody binding:

  • Verify that disease mutations don't alter the antibody epitope (aa 1308-1455)

  • Compare antibody affinity between wild-type and mutant proteins using titration experiments

  • Consider using alternative antibodies targeting different epitopes as controls

• Quantification of mutant phenotypes:

  • Measure changes in condensate formation (number, size, morphology)

  • Quantify partition coefficients to assess enrichment efficiency

  • Analyze differences in recruitment of binding partners (Dendrin, CD2AP, Nephrin)

  • Compare FRAP recovery parameters to detect changes in material properties

• Experimental design for mutation studies:

  • Generate paired cell lines with isogenic backgrounds (CRISPR knock-in of mutations)

  • Use transfected wild-type and mutant constructs at matched expression levels

  • Include heterozygous conditions to model carrier states

  • Perform rescue experiments to confirm mutation-specific effects

• Advanced analytical approaches:

  • Single-molecule tracking to detect changes in MAGI2 dynamics

  • FLIM-FRET to measure interaction strengths between mutant MAGI2 and binding partners

  • Correlative light-electron microscopy to link fluorescence patterns to ultrastructural features

  • Optogenetic perturbation of phase separation to assess dynamic responses

• Disease-relevant conditions:

  • Test phase separation under disease-mimicking conditions (e.g., altered calcium levels, oxidative stress)

  • Examine effects of nephrotoxic drugs on wild-type versus mutant MAGI2 condensates

  • Study condensate properties in patient-derived podocytes or kidney organoids

• Comparative mutational analysis recommended workflow:

  • Express GFP-tagged wild-type and mutant MAGI2 in relevant cell types

  • Fix and immunostain with FITC-conjugated MAGI2 antibodies and markers for binding partners

  • Quantify condensate properties and colocalization parameters

  • Perform FRAP analysis to assess molecular dynamics

  • Correlate molecular findings with functional readouts (e.g., podocyte morphology, filtration)

These approaches enable detailed characterization of how disease-associated mutations affect MAGI2's phase separation properties and interactions, potentially revealing mechanisms of pathogenesis and therapeutic targets.