MICAL1 Antibody

What is MICAL1 and why is it significant for research?

MICAL1 (microtubule associated monoxygenase, calponin and LIM domain containing 1) is a multidomain flavoprotein monooxygenase with diverse cellular functions. It has four conserved domains: an N-terminal flavin adenine dinucleotide (FAD) binding domain, a calponin homology (CH) domain, a Lin11, Isl-1 and Mec-3 (LIM) domain, and a C-terminal coiled-coil (CC) domain . MICAL1 regulates actin stress fibers and is required for normal actin organization by promoting depolymerization of F-actin through oxidation of specific methionine residues. It may also be involved in apoptosis through binding with NDR (nuclear Dbf2-related) kinases . Recent research has implicated MICAL1 in cancer progression, cell migration, and viral processes, making it an important target for antibody-based studies.

What types of MICAL1 antibodies are available and what are their primary applications?

MICAL1 antibodies are available in both monoclonal and polyclonal forms, with various host species including mouse and rabbit. The primary applications include:

These antibodies have been validated in multiple species, with most showing reactivity to human and mouse MICAL1, while some also react with rat samples .

What is the optimal storage condition for MICAL1 antibodies?

Storage conditions vary depending on the specific antibody formulation:

• Unconjugated antibodies in PBS only should be stored at -80°C for maximum stability

• Antibodies in PBS with 0.02% sodium azide and 50% glycerol should be stored at -20°C for long-term storage

• For frequent use over short periods (up to one month), 4°C storage may be suitable for some formulations

• Avoid repeated freeze-thaw cycles as they can compromise antibody quality and performance

Always refer to the manufacturer's specific recommendations for your particular antibody.

How should I optimize Western blot conditions for detecting MICAL1?

When performing Western blot analysis for MICAL1:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent degradation

• Gel selection: Since MICAL1 has a high molecular weight (~118-120 kDa), use lower percentage gels (8-10% SDS-PAGE) for better resolution

• Transfer conditions: Longer transfer times or higher voltage may be necessary for efficient transfer of this high molecular weight protein

• Blocking: 5% non-fat milk or BSA in TBST is typically effective

• Antibody dilution: Start with manufacturer's recommended dilution (often 1:2000-1:16000) , but optimize as needed

• Positive controls: Include lysates from cells known to express MICAL1 (e.g., Jurkat cells, HEK-293 cells, T-47D cells, HeLa cells)

• Detection: Enhanced chemiluminescence (ECL) is usually sufficient, but for lower expression levels, more sensitive detection systems may be required

Importantly, the observed molecular weight of MICAL1 is typically around 120 kDa, slightly higher than the calculated 118 kDa , which is common for many proteins due to post-translational modifications.

What are the best techniques for validating MICAL1 antibody specificity?

Proper validation of MICAL1 antibody specificity is critical for reliable results:

• Positive and negative controls:

  • Use cell lines with documented MICAL1 expression (e.g., HeLa, Jurkat, T-47D) as positive controls

  • Include tissues known to express or lack MICAL1

  • Use siRNA/shRNA knockdown or CRISPR knockout samples as negative controls

• Multiple detection methods:

  • Compare results across different applications (WB, IHC, IF) to ensure consistent detection patterns

  • Use antibodies targeting different epitopes of MICAL1 to confirm specificity

• Immunogen competition:

  • Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction

  • Some manufacturers offer blocking peptides specifically for this purpose

• Multiple antibody validation:

  • Compare results using antibodies from different sources or clones

  • Look for consistent results across monoclonal and polyclonal antibodies

• Molecular weight verification:

  • Confirm that the detected band matches the expected molecular weight (~120 kDa)

  • Be aware of potential splice variants or post-translational modifications

How can I effectively design experiments to study MICAL1 interactions with binding partners?

To investigate MICAL1 protein-protein interactions:

• Co-immunoprecipitation:

  • Use anti-MICAL1 antibodies for IP followed by Western blotting for suspected binding partners

  • Alternatively, IP the suspected partner and blot for MICAL1

  • Include appropriate controls (IgG, lysate inputs)

  • Consider crosslinking for transient interactions

• Proximity ligation assay (PLA):

  • Useful for detecting proteins that interact in situ

  • Requires antibodies raised in different species

  • Provides spatial information about interactions

• Pull-down assays:

  • Use recombinant MICAL1 domains to identify specific interaction regions

  • For example, the CC domain of MICAL1 has been shown to interact with MyoVa

• FRET/BRET approaches:

  • For real-time interaction studies in living cells

  • Requires fluorescent/luminescent protein tagging

• Yeast two-hybrid screening:

  • For discovery of novel interaction partners

  • Follow up with biochemical validation

When studying MICAL1 interactions, consider its domain structure and known binding regions. For example, MICAL1 binds to MyoVa through a GTBM (GTD-binding motif), which is present in MICAL1 but not in MICAL2 or MICAL3 .

How can I effectively investigate MICAL1's role in actin dynamics using antibody-based approaches?

MICAL1 promotes F-actin disassembly through oxidation of specific methionine residues. To study this function:

• Immunofluorescence co-localization:

  • Use anti-MICAL1 antibodies alongside F-actin staining (phalloidin)

  • Analyze co-localization patterns in various cellular contexts

  • Time-course experiments can reveal dynamics of recruitment

• Live-cell imaging with MICAL1 knockdown/rescue:

  • Deplete endogenous MICAL1 using siRNA or CRISPR

  • Rescue with wild-type or mutant MICAL1 (e.g., MICAL1-3G3W catalytically inactive mutant)

  • Monitor actin dynamics with fluorescent actin probes

• In vitro actin disassembly assays:

  • Immunoprecipitate MICAL1 from cells under different conditions

  • Test activity on purified F-actin

  • Compare with recombinant MICAL1

• F-actin quantification following manipulation:

  • Use IF or flow cytometry to quantify F-actin levels

  • Compare control vs. MICAL1-depleted cells

  • Rescue experiments with domain mutants (e.g., MICAL1ΔGTBM)

Research has demonstrated that MICAL1 knockdown significantly increases F-actin levels at structures like the midbody during cytokinesis, while overexpression of wild-type MICAL1 (but not catalytically inactive MICAL1-3G3W) reduces F-actin levels .

What approaches should I use to study MICAL1's activation mechanisms and autoinhibition?

MICAL1 exists in an autoinhibited state that can be activated by various mechanisms:

• Domain deletion/mutation analysis:

  • Create constructs lacking specific domains (MO, CH, LIM, CC)

  • Test activity using actin depolymerization assays

  • Use antibodies specific to different domains to track conformational changes

• Study of activator interactions:

  • RAB proteins (particularly RAB35) can activate MICAL1

  • PAK1 has been identified as an activator of MICAL1

  • Examine co-localization and binding under activating conditions

• Structural studies supported by antibody validation:

  • Recent cryo-EM structures show that MICAL1 autoinhibition involves the binding of CH and LIM domains to the CC domain

  • This maintains the CC domain in a conformation that interacts with the catalytic domain

  • Use domain-specific antibodies to track conformational changes upon activation

• Phosphorylation analysis:

  • Evidence suggests MICAL1 activity may be regulated by phosphorylation

  • Use phospho-specific antibodies if available, or general phospho-antibodies after IP

  • Mass spectrometry analysis of immunoprecipitated MICAL1 can identify modification sites

Recent research indicates that "MICAL1 autoinhibition hinges on the binding of the CH and LIM domains, facilitated by a helical region of the long linker, to the CC domain" . During Rab-induced activation, Rab binding to the CC domain likely triggers conformational changes that destabilize this autoinhibitory interaction.

What are the recommended approaches for studying MICAL1's role in cancer progression?

MICAL1 has been implicated in multiple cancer types, including pancreatic cancer and renal clear cell carcinoma. To investigate its role:

• Expression analysis in patient samples:

  • IHC staining of tissue microarrays with anti-MICAL1 antibodies

  • Compare expression between tumor and adjacent normal tissues

  • Correlate with clinical parameters and survival data

• Functional studies in cancer cell lines:

  • Generate stable MICAL1-overexpressing or MICAL1-silencing cells

  • Assess effects on:

    • Proliferation (CCK-8, colony formation, EdU assays)

    • Migration and invasion (wound healing, Transwell assays)

    • In vivo tumor growth and metastasis models

• Mechanism investigation:

  • Examine effects on signaling pathways (e.g., WNT/β-catenin)

  • Study interaction with specific proteins related to cancer progression

  • Assess effects on cytoskeletal reorganization during invasion

• Therapeutic targeting:

  • Test effects of inhibiting MICAL1 on cancer cell sensitivity to treatments

  • Explore combination approaches

  • Develop screening assays for MICAL1 inhibitors

Why might I observe multiple bands when using MICAL1 antibodies in Western blot?

Multiple bands in MICAL1 Western blots could result from:

• Alternative splicing:

  • MICAL1 may have splice variants

  • Consult databases for known splice variants and their predicted molecular weights

  • Use antibodies targeting different epitopes to confirm identity

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or other modifications can alter mobility

  • Use phosphatase treatment to test for phosphorylation

  • Mass spectrometry analysis can identify modifications

• Proteolytic degradation:

  • MICAL1 may be sensitive to proteolysis during sample preparation

  • Use fresh samples with complete protease inhibitor cocktails

  • Optimize lysis and sample handling conditions

• Cross-reactivity:

  • Antibodies may recognize related proteins (MICAL2, MICAL3, MICAL-L1, MICAL-L2)

  • Validate using knockout/knockdown controls

  • Compare results with different antibodies

• Non-specific binding:

  • Optimize blocking conditions and antibody dilutions

  • Try different blocking agents (milk vs. BSA)

  • Increase washing stringency

The full-length MICAL1 protein is expected at ~120 kDa, with potential fragments at lower molecular weights depending on the epitope recognized by your antibody.

How can I resolve discrepancies between different MICAL1 antibodies in my experimental results?

When facing inconsistent results between different MICAL1 antibodies:

• Epitope mapping:

  • Determine the epitope regions recognized by each antibody

  • Different domains may have different accessibility in various experimental conditions

  • Some antibodies target N-terminal regions (e.g., FAD domain) while others target C-terminal regions (e.g., CC domain)

• Validation strategy:

  • Use MICAL1 knockdown/knockout samples as negative controls

  • Test all antibodies against the same positive and negative controls

  • Include recombinant MICAL1 as a standard when possible

• Application optimization:

  • Each antibody may require specific optimization for different applications

  • Adjust fixation methods for IF/IHC (aldehyde vs. organic solvent)

  • Try different antigen retrieval methods for IHC

  • Modify blocking and incubation conditions

• Conformational considerations:

  • MICAL1 has an autoinhibited conformation where some epitopes may be masked

  • Activation (e.g., by RAB proteins) may expose additional epitopes

  • Denaturing vs. native conditions may affect epitope accessibility

• Cross-validation approaches:

  • Use alternative detection methods (e.g., mass spectrometry)

  • Tag endogenous MICAL1 using CRISPR knock-in strategies

  • Employ proximity labeling approaches

For example, when studying MICAL1's role in cytokinesis, researchers validated their findings using both siRNA knockdown and rescue experiments with siRNA-resistant MICAL1 constructs .

How should I interpret MICAL1 localization patterns in immunofluorescence studies?

MICAL1 shows distinct localization patterns depending on cellular context:

• Typical localization patterns:

  • Cytoplasmic distribution with possible enrichment at specific structures

  • Accumulation at the midbody during cytokinesis

  • Potential association with actin structures

  • Co-localization with binding partners like MyoVa or RAB proteins

• Factors affecting localization:

  • Cell type and physiological state

  • Cell cycle stage (particularly evident during cytokinesis)

  • Activation status of MICAL1

  • Fixation and permeabilization methods

• Validation approaches:

  • Compare multiple antibodies targeting different epitopes

  • Use tagged MICAL1 constructs (being careful about potential artifacts)

  • Include domain deletion mutants to map localization determinants

  • Perform co-localization with known markers and partners

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed localization studies

  • Live-cell imaging with fluorescently tagged MICAL1

  • FRAP (Fluorescence Recovery After Photobleaching) for dynamics studies

Research has shown that "MICAL1 does not directly interact with Rab11a" but "drives the accumulation of Rab11a-positive vesicles at the midbody by binding to MyoVa" , highlighting the importance of careful interpretation of co-localization data.

How can MICAL1 antibodies be used to study its role in viral infection processes?

Recent research has implicated MICAL1 in viral infection processes, particularly in HIV-1 budding:

• Virus budding studies:

  • Use MICAL1 antibodies to track localization during viral budding

  • Compare infected vs. uninfected cells

  • Co-localization with viral components

  • Super-resolution microscopy to visualize structural details

• Mechanistic investigations:

  • MICAL1 locally depolymerizes actin at budding sites to promote HIV-1 budding and release

  • Upon MICAL1 depletion, F-actin abnormally remains at viral budding sites

  • Study the recruitment of ESCRT (Endosomal Sorting Complexes Required for Transport) machinery

• RAB35-MICAL1 pathway:

  • RAB35 acts as a MICAL1 activator and is recruited to budding sites

  • Use antibodies to track both proteins during infection

  • Examine the coordination between these factors

• Branched actin networks:

  • MICAL1 directly disassembles branched-actin networks

  • Study interaction with Arp2/3 complex components

  • Investigate effects of Arp2/3 inhibitors in MICAL1-depleted cells

Research shows that "viral release can be restored in MICAL1-depleted cells by inhibiting Arp2/3-dependent branched actin networks" , suggesting a specific role in remodeling branched actin during viral budding.

What approaches should be used to investigate MICAL1's role in immune cell function?

MICAL1's role in immune responses is an emerging area of research:

• Expression analysis in immune cell subsets:

  • Use flow cytometry with anti-MICAL1 antibodies to profile expression across immune cell types

  • Examine changes in expression upon activation/differentiation

  • Compare expression in healthy vs. disease states

• T cell exhaustion studies:

  • MICAL1 expression has been positively associated with T cell exhaustion markers

  • Investigate co-expression with PD-1, CTLA-4, LAG-3, etc.

  • Study functional consequences of MICAL1 modulation on T cell activity

• Immune infiltration analysis:

  • MICAL1 expression in tumors positively correlates with CD8+/Treg cell infiltration levels

  • Use multiplex IHC to study co-localization in tumor microenvironment

  • Functional studies in relevant immune cell models

• Signaling pathway analysis:

  • KEGG and GSEA analyses suggest MICAL1 is involved in immune-related pathways

  • Study effects of MICAL1 modulation on immune signaling pathways

  • Investigate intersection with cytoskeletal regulation during immune cell function

Bioinformatic analyses have revealed that "MICAL1 expression had strong relationships with various T cell exhaustion markers" and is "positively associated with CD8+/Treg cell infiltration levels" , suggesting important but not fully characterized roles in immune function.

How can phospho-specific or conformation-specific MICAL1 antibodies advance our understanding of its regulation?

Development and application of specialized MICAL1 antibodies could provide unprecedented insights:

• Phospho-specific antibodies:

  • MICAL1 activation by PAK1 suggests phosphorylation-based regulation

  • Identify key phosphorylation sites through mass spectrometry

  • Develop antibodies specific to these phospho-epitopes

  • Use to track activation status in different cellular contexts

• Conformation-specific antibodies:

  • MICAL1 undergoes significant conformational changes during activation

  • Antibodies recognizing active vs. inactive conformations could track activation in situ

  • Design immunogens that stabilize specific conformational states

  • Validate using mutants locked in specific conformations

• Domain-specific antibodies:

  • Target individual domains (MO, CH, LIM, CC) to study domain-specific functions

  • Use to track domain accessibility during activation/inactivation

  • Combine with functional studies using domain deletion/mutation constructs

• Applications in high-throughput screening:

  • Develop ELISA or flow cytometry-based assays using conformation-specific antibodies

  • Screen for compounds that modulate MICAL1 activation

  • Potential therapeutic applications in cancer or viral infections

The recent cryo-EM structure showing that "MICAL1 autoinhibition hinges on the binding of the CH and LIM domains... to the CC domain" provides structural insights that could guide the development of such specialized antibodies.