mical3a Antibody

What is MICAL3 and what are its key structural domains?

MICAL3 (Microtubule Associated Monooxygenase, Calponin and LIM domain containing 3) is a multidomain protein with a mass of approximately 224.3 kDa and 2002 amino acid residues in humans. It contains a flavoprotein monooxygenase domain, a calponin-homology domain, and a LIM zinc-binding domain. These structural components are essential for its diverse cellular functions, with the monooxygenase domain utilizing flavin adenine dinucleotide (FAD) as a cofactor. The protein has nuclear and cytoplasmic localization and exists in up to five different isoforms .

What are the primary cellular functions of MICAL3?

MICAL3 functions as an F-actin disassembly factor through its monooxygenase activity, which mediates oxidation of specific methionine residues on actin to form methionine-sulfoxide, preventing repolymerization. Beyond actin regulation, MICAL3 plays critical roles in:

• Cytokinesis - targeting adaptor protein ELKS and Rab8A-positive vesicles to the midbody during cell division

• Vesicle trafficking - interacting with Rab proteins for vesicle targeting

• Semaphorin signaling - mediating semaphorin-induced signal transduction in neurons and cancer cells

• Scaffolding - serving as a midbody-associated scaffold during abscission

How should researchers select the appropriate MICAL3 antibody for their specific application?

When selecting a MICAL3 antibody, researchers should consider:

• Target region specificity: Determine whether you need antibodies targeting specific domains (e.g., monooxygenase domain versus C-terminal region)

• Application compatibility: Verify validated applications (WB, IF, ICC, IP, ELISA) for your planned experiments

• Species reactivity: Confirm cross-reactivity with your model organism (human, mouse, rat, etc.)

• Clonality: Consider whether monoclonal antibodies (like clone 3A6, M3, or 30A10) or polyclonal antibodies better suit your research needs

• Validation evidence: Review published literature using the antibody and examine vendor validation data

For detection of specific interactions, such as MICAL3-MKLP1 binding in cytokinesis studies, antibodies with validated performance in proximity ligation assays may be required .

What methodologies are used to validate MICAL3 antibodies?

Validation of MICAL3 antibodies typically follows these methodological approaches:

• Specificity testing:

  • Western blotting against cell lysates with endogenous MICAL3 expression

  • Testing in MICAL3 knockout models (CRISPR/Cas9-generated cell lines)

  • Peptide competition assays

• Application-specific validation:

  • Immunocytochemistry: Verification of expected subcellular localization (midbody during cytokinesis, cytoplasmic distribution)

  • Immunoprecipitation: Confirmation of known protein interactions (e.g., ELKS, Rab8A)

  • Cross-reactivity assessment: Testing against related MICAL family proteins

• Technical performance criteria:

  • Signal-to-noise ratio determination

  • Titration experiments to establish optimal working concentrations

  • Reproducibility assessment across different protein lots or tissue samples

How can MICAL3 antibodies be optimized for detecting its localization during cytokinesis?

For optimal detection of MICAL3 during cytokinesis:

• Fixation method selection:

  • Paraformaldehyde (4%) fixation followed by Triton X-100 permeabilization preserves midbody structures

  • Methanol fixation may be preferable for certain epitopes but can disrupt membrane structures

• Co-staining strategy:

  • Pair MICAL3 antibodies with antibodies against known binding partners such as MKLP1, which serves as a midbody marker

  • Include tubulin staining to visualize the central spindle and midbody

• Time-course imaging:

  • Capture images from anaphase through abscission, as MICAL3 shows "conspicuous localization at the spindle midzone starting from anaphase and strongly accumulated at the central spindle and the midbody during cytokinesis"

• Protocol optimization:

  • Use 1:100 to 1:500 dilution of primary antibody (depending on specific antibody concentration)

  • Employ high-sensitivity detection methods for lower-expressed MICAL3 variants

  • Consider confocal microscopy for precise localization analysis

What are the recommended protocols for using MICAL3 antibodies in Western blotting?

For optimal Western blotting results with MICAL3 antibodies:

Protocol Recommendations:

• Sample preparation:

  • Lyse cells in buffer containing protease inhibitors

  • Use lower percentage (6-8%) SDS-PAGE gels due to MICAL3's large size (224.3 kDa)

• Blocking conditions:

  • Block membranes in 5% BSA or milk with 0.05% Tween-20 for 1 hour at room temperature

• Antibody incubation:

  • Primary antibody: Dilute in 5% BSA or milk and incubate for 1 hour at room temperature or overnight at 2-8°C

  • Secondary antibody: Incubate in 5% milk for 1 hour at room temperature

• Detection optimization:

  • For full-length MICAL3: Enhanced chemiluminescence (ECL) detection

  • For detecting multiple isoforms: Consider using gradient gels

  • When studying dimerization: Use non-reducing SDS-PAGE conditions (as demonstrated for CRMP2 dimerization studies)

• Troubleshooting:

  • For weak signals: Extend primary antibody incubation to overnight at 4°C

  • For high background: Increase washing duration and number of washes

  • For detection of post-translational modifications: Use phospho-specific antibodies alongside total MICAL3 antibodies

How can MICAL3 antibodies be used to study protein-protein interactions during cytokinesis?

To investigate MICAL3's protein interactions during cytokinesis:

• Proximity Ligation Assay (PLA) approach:

  • PLA can detect protein-protein interactions with high sensitivity (as demonstrated for MICAL3-CRMP2 interactions)

  • Combine MICAL3 antibody with antibodies against suspected binding partners (MKLP1, ELKS, Rab8A)

  • Quantify interaction by counting fluorescent PLA dots in midbody regions

• Co-immunoprecipitation strategy:

  • Use MICAL3 antibodies for pull-down experiments followed by immunoblotting for interacting partners

  • For enhanced detection, consider using cross-linking approaches as employed in the MICAL3-MKLP1 interaction studies

• Mass spectrometry pipeline:

  • Implement bioGFP-tagged MICAL3 with streptavidin pulldown followed by mass spectrometry (similar to methods that identified ELKS and Rab8A as MICAL3 partners)

  • Validate identified interactions using MICAL3 antibodies in reverse immunoprecipitation

  • Analyze data using specialized interaction proteomics software

• Relevant research findings:
  MICAL3 forms complexes with multiple proteins during cytokinesis:

  • MICAL3-MKLP1 interaction occurs directly via the C-terminus of MICAL3

  • MICAL3-ELKS interaction occurs independently of its enzymatic activity

  • MICAL3 can form a triple complex with MKLP1 and ELKS, connecting ELKS to the midbody

What approaches combine MICAL3 antibodies with genetic manipulation to understand its function?

Integrating MICAL3 antibodies with genetic approaches provides powerful insights:

• CRISPR/Cas9 knockout validation:

  • MICAL3 antibodies can confirm complete protein loss in knockout cells

  • In published research, CRISPR/Cas9-mediated MICAL3 knockout cells showed:

    • Delayed abscission (>2-fold increase in time between furrow ingression and abscission)

    • Increased cytokinetic failure (~10% binucleated cells vs. ~2% in controls)

    • Loss of ELKS localization at midbodies

    • Reduced Rab8A-positive structures at intercellular bridges

• Rescue experiment design:

  • Express wild-type or mutant MICAL3 constructs in knockout cells

  • Use antibodies to detect localization patterns and restoration of protein-protein interactions

  • Published findings showed that enzymatically dead MICAL3 mutants could rescue cytokinetic defects, indicating enzymatic activity is dispensable for this function

• Domain-specific mutation analysis:

  • Create targeted mutations in specific MICAL3 domains

  • Use antibodies to assess effects on:

    • Protein stability and expression

    • Localization to midbody structures

    • Interaction with binding partners

  • For example, deletion of the MKLP1-binding C-terminus prevented rescue of the binucleated phenotype

How can MICAL3 antibodies be applied in the study of cancer biology, particularly regarding breast cancer stem cells?

MICAL3 antibodies offer valuable tools for investigating cancer biology:

• Cell pair assay methodology:

  • Antibodies can be used to track symmetrical vs. asymmetrical division of cancer stem cells

  • In breast cancer studies, NP1-positive cells were tracked for division patterns using immunostaining

  • MICAL3 depletion significantly decreased symmetric cell division induced by Sema3A

• Cancer stem cell identification:

  • Using MICAL3 antibodies alongside stem cell markers (NP1, Numb) can help identify and track cancer stem cell populations

  • Research showed that Numb proteins are stabilized in NP1-positive cancer stem cells but not in differentiated cells

• Prognostic biomarker evaluation:

  • Tissue microarray analysis with MICAL3 antibodies can be used to assess correlation with clinical outcomes

  • Expression levels of both MICAL3 and Sema3A showed significantly worse outcomes in breast cancer patients

• Therapeutic target validation:

  • Similar to approaches used with MICA/MICB antibodies, MICAL3 antibodies could potentially be used to develop therapeutic strategies

  • Targeting MICAL3 function in cancer cells could reduce symmetric division of cancer stem cells, potentially reducing tumor growth

What are common challenges and solutions when using MICAL3 antibodies in immunofluorescence applications?

Researchers face several challenges when using MICAL3 antibodies for immunofluorescence:

For specific immunofluorescence applications:

• When studying MICAL3 at the midbody, co-stain with α-tubulin to visualize the central spindle structure

• For tracking MICAL3 throughout cell division, combine with live imaging of fluorescently tagged markers

How should researchers interpret contradictory results when using different MICAL3 antibodies?

When facing contradictory results with different MICAL3 antibodies:

• Epitope mapping analysis:

  • Determine target regions of each antibody (N-terminal, monooxygenase domain, C-terminal)

  • Different domains may be masked in certain protein complexes or subject to post-translational modifications

  • Some antibodies may recognize specific conformational states of MICAL3

• Isoform recognition profile:

  • Verify which of the five reported MICAL3 isoforms each antibody recognizes

  • Different antibodies may preferentially detect certain isoforms, leading to apparent discrepancies

• Validation using genetic approaches:

  • Test antibodies in MICAL3 knockout or knockdown systems as negative controls

  • Use overexpression systems with tagged MICAL3 constructs to confirm specificity

  • Consider domain-specific deletions to map exact recognition sites

• Application-specific considerations:

  • An antibody performing well in Western blotting may fail in immunoprecipitation or immunofluorescence

  • Fixation methods may differently affect epitope accessibility

  • When studying interactions like MICAL3-MKLP1 binding, the interaction itself may mask certain epitopes

How can MICAL3 antibodies be integrated with super-resolution microscopy techniques?

MICAL3 antibodies can be leveraged with advanced microscopy through:

• STED microscopy applications:

  • Employ fluorophore-conjugated secondary antibodies optimized for STED

  • Achieve sub-diffraction resolution of MICAL3 localization at the midbody (typically 70-90 nm)

  • Enable precise co-localization analysis with binding partners such as MKLP1 and ELKS

• STORM/PALM microscopy implementation:

  • Use photoswitchable fluorophore-conjugated antibodies

  • Map the nanoscale organization of MICAL3 at the intercellular bridge

  • Quantify molecular clustering patterns during different stages of cytokinesis

• Expansion microscopy approach:

  • Physically expand fixed samples using polymer networks

  • Maintain antibody labeling through the expansion process

  • Visualize detailed MICAL3 distribution with conventional microscopes

• Live-cell super-resolution imaging:

  • Combine with Fab fragments of MICAL3 antibodies for less invasive live imaging

  • Track dynamic MICAL3 recruitment during midbody maturation and abscission

What are the methodological considerations for using MICAL3 antibodies in in vivo research models?

For in vivo applications of MICAL3 antibodies:

• Tissue penetration optimization:

  • Consider using Fab fragments or smaller antibody formats for better tissue penetration

  • Employ appropriate antigen retrieval methods for fixed tissues

  • For thick sections, extend antibody incubation times or use specialized clearing techniques

• In vivo imaging approaches:

  • Directly labeled MICAL3 antibodies can be used for intravital microscopy

  • Consider near-infrared fluorophore conjugates for deeper tissue imaging

  • For longer-term studies, develop stable cell lines expressing fluorescent-tagged MICAL3

• Animal model considerations:

  • Validate species cross-reactivity before in vivo applications

  • Available MICAL3 antibodies show reactivity with mouse, rat, and human proteins

  • For humanized mouse models studying cancer metastasis (similar to approaches used with MICA/MICB antibodies), confirm antibody specificity for human MICAL3

• Developmental studies:

  • MICAL3's role in semaphorin signaling makes it relevant for neuronal development studies

  • Consider timing of antibody administration in developing animal models

  • Pair with lineage tracing approaches for complete developmental analysis

What novel antibody-based approaches could advance understanding of MICAL3's role in health and disease?

Innovative antibody approaches for MICAL3 research include:

• Conformation-specific antibodies:

  • Develop antibodies that specifically recognize active versus inactive MICAL3 conformations

  • Create antibodies that detect specific post-translational modifications (phosphorylation, oxidation)

  • Generate antibodies that distinguish between different oligomeric states

• Bi-specific antibody applications:

  • Engineer bi-specific antibodies targeting MICAL3 and interacting partners

  • Use such tools to investigate transient complexes during cytokinesis

  • Apply similar approaches to study MICAL3 in cancer progression, as demonstrated with other therapeutic antibodies

• Intrabody development:

  • Create genetically encoded antibody fragments expressed within cells

  • Target specific domains to inhibit particular MICAL3 functions without complete protein elimination

  • Combine with inducible expression systems for temporal control

• Therapeutic potential exploration:

  • Similar to antibody approaches targeting MICA/MICB in cancer, develop strategies targeting MICAL3

  • In breast cancer research, MICAL3 has been implicated in cancer stem cell symmetrical division

  • Investigate whether MICAL3-blocking antibodies could inhibit cancer stem cell proliferation

How might advances in antibody engineering enhance MICAL3 research tools?

Emerging antibody technologies applicable to MICAL3 research:

• Domain-specific nanobodies:

  • Develop small single-domain antibodies (~15 kDa) against specific MICAL3 regions

  • Advantages include smaller size for improved penetration and reduced interference

  • Potential applications in super-resolution microscopy and live-cell imaging

• Site-specific conjugation techniques:

  • Employ enzymatic or chemical approaches for precise fluorophore attachment

  • Create homogeneous antibody reagents with consistent labeling ratios

  • Enhance quantitative applications by ensuring uniform fluorescent properties

• Recombinant antibody fragments:

  • Engineer Fab, scFv, or diabody formats for improved tissue penetration

  • Develop recombinant antibodies with enhanced specificity for individual MICAL3 isoforms

  • Create stable, reproducible reagents to address batch-to-batch variation issues

• Genetically encoded probes:

  • Create split-GFP complementation systems based on MICAL3 antibody binding

  • Develop FRET-based sensors incorporating antibody-derived binding domains

  • Establish optogenetic tools that incorporate MICAL3-targeting domains