mical3b Antibody

What is MICAL3 and why is it significant in research?

MICAL3 is a multidomain protein that functions in vesicle trafficking, cytoskeletal organization, and cell signaling pathways. It contains an N-terminal monooxygenase (MO) domain with FAD-binding activity that is critical for its function in oxidizing proteins and mediating protein-protein interactions . Research has shown that MICAL3 plays a significant role in the Semaphorin signaling pathway, where it mediates interactions between key proteins like CRMP2 and Numb . In cancer biology, MICAL3 has been implicated in controlling symmetric versus asymmetric cell division of cancer stem cells, with higher expression levels associated with worse outcomes in breast cancer patients .

What are the essential domains of MICAL3 relevant for antibody targeting?

MICAL3 contains several functional domains that can be targeted by antibodies:

Antibodies targeting specific domains can help distinguish between different functions of MICAL3. The coiled-coil regions have been shown to be particularly important for interactions with proteins like ELKS as demonstrated in truncation studies .

What are the standard applications for MICAL3 antibodies in research?

MICAL3 antibodies can be utilized across multiple experimental approaches:

• Western Blotting (WB): For detecting MICAL3 expression levels in cell or tissue lysates

• Immunoprecipitation (IP): For isolating MICAL3 and its binding partners

• ELISA: For quantitative determination of MICAL3 levels

• Proximity Ligation Assay (PLA): For detecting in situ protein-protein interactions, as demonstrated in studies of CRMP2-Numb interactions mediated by MICAL3

• Immunofluorescence: For studying subcellular localization and distribution during cell division

How can researchers validate MICAL3 antibody specificity?

Rigorous validation of MICAL3 antibodies is essential for experimental reliability:

• Gene knockdown/knockout controls: Test antibody reactivity in MICAL3-depleted cells using siRNA or shRNA approaches as demonstrated in studies of MICAL3 knockdown effects on Sema3A signaling

• Domain mapping validation: Use truncation mutants of MICAL3 to confirm epitope specificity, similar to the approach used in characterizing MICAL3 interaction domains

• Cross-reactivity assessment: Test against related family proteins and across species if conducting comparative studies

• Multiple detection methods: Confirm antibody specificity using different applications (WB, IP, IF) to ensure consistent results

How do MICAL3 antibodies contribute to cancer stem cell research?

MICAL3 antibodies have proven instrumental in elucidating cancer stem cell biology:

Breast cancer studies have shown that MICAL3 is involved in the Sema3A/NP1/MICAL3/CRMP2/Numb signaling pathway that induces symmetric division of breast cancer stem cells (BCSCs) . Using MICAL3 antibodies in combination with cell pair assays, researchers have been able to track the distribution of MICAL3 during symmetric versus asymmetric cell division .

Key findings include:

• Breast cancer patients with higher levels of MICAL3 expression showed worse outcomes than those with lower expression levels

• MICAL3 knockdown significantly decreased symmetric cell division in Sema3A-containing medium

• MICAL3's MO domain is essential for the interaction between CRMP2 and Numb proteins in the presence of Sema3A

What methodologies can be used to study MICAL3's role in the Semaphorin signaling pathway?

The Semaphorin signaling pathway can be investigated using MICAL3 antibodies through several methodologies:

• Protein-Protein Interaction Analysis:

  • Use proximity ligation assays (PLA) with MICAL3 antibodies to detect interactions with pathway components

  • The number of PLA dots indicating CRMP2-Numb interaction was greatly increased in Sema3A-containing medium but decreased with MICAL3 knockdown

• Functional Domain Analysis:

  • Transfect cells with constructs expressing wild-type MICAL3 (MICAL3-WT) or mutant MICAL3 (MICAL3-3G3W) to determine domain functionality

  • PLA dots were observed much more frequently in MICAL3-WT-transfected cells than in MICAL3-3G3W-transfected cells, indicating the importance of the MO domain

• Protein Accumulation Studies:

  • Use Western blotting with MICAL3 antibodies to monitor changes in protein levels

  • Stimulation with Sema3A increased Numb protein in breast cancer cells, while MICAL3 knockdown inhibited this accumulation

How can cross-linking mass spectrometry (XL-MS) be combined with MICAL3 antibodies for interaction mapping?

Cross-linking mass spectrometry (XL-MS) combined with MICAL3 antibodies provides a powerful approach for mapping protein interaction domains:

• Research has demonstrated that XL-MS can be used to identify cross-linked lysine residues between MICAL3 and its interaction partners like ELKS and Rab8A

• Based on XL-MS data, truncation mutants of MICAL3 can be designed to include varying numbers of the cross-linked lysine residues

• Pull-down assays using these truncation mutants can then determine the minimal regions sufficient for protein interactions:

  • For MICAL3-ELKS interaction, the shortest construct sufficient (MICAL3-C5) encompassed a single coiled-coil region containing two lysine residues involved in cross-linking

  • Similar approaches identified regions important for MICAL3-Rab8A interaction

This methodology allows precise mapping of interaction domains without relying on predicted domain boundaries, providing more accurate information for antibody design and functional studies.

What are the technical considerations for co-immunoprecipitation studies using MICAL3 antibodies?

When conducting co-immunoprecipitation studies with MICAL3 antibodies, researchers should consider:

• Antibody Selection: Choose antibodies validated for IP applications like the Mouse Anti-MICAL3 Recombinant Antibody (clone 30A10) , which offers:

  • Increased sensitivity

  • Confirmed specificity

  • High repeatability

  • Excellent batch-to-batch consistency

• Buffer Optimization: Different buffer compositions can significantly affect the stability of protein complexes:

  • For studying MICAL3-ELKS interactions, buffers that preserve coiled-coil interactions would be optimal

  • For MICAL3-Rab8A interactions, consider buffers compatible with GTPase activity

• Validation Controls: Include appropriate controls:

  • MICAL3 knockdown or knockout samples as negative controls

  • Truncation mutants to confirm domain-specific interactions

  • IgG controls to account for non-specific binding

How can MICAL3 antibodies be used to investigate therapeutic strategies targeting cancer stem cells?

Recent research suggests MICAL3 as a potential therapeutic target, particularly in cancers where stem cell dynamics play a crucial role:

• MICAL3 antibodies can be used to:

  • Monitor changes in MICAL3 expression following treatment with experimental compounds

  • Track alterations in symmetric/asymmetric division patterns of cancer stem cells

  • Evaluate effects of MICAL3 inhibition on tumor sphere formation

• Similar approaches have been demonstrated with other therapeutic antibodies:

  • The novel MICA/B antibody AHA-1031 shows how targeting specific protein domains can have therapeutic effects in cancer models

  • In KL mutant NSCLC xenograft models, treatment with AHA-1031 significantly inhibited tumor growth compared with vehicle-treated animals

What methods can be used to study the redox activity of MICAL3's MO domain?

The monooxygenase (MO) domain of MICAL3 contains redox activity that can be studied using specialized approaches:

• Activity Assays:

  • Measure NADPH consumption rates in the presence of purified MICAL3 or MICAL3 fragments containing the MO domain

  • Compare wild-type MICAL3 with mutants like MICAL3-3G3W that affect the MO domain function

• Redox-Sensitive Probes:

  • Use redox-sensitive fluorescent proteins to detect MICAL3-induced oxidation in living cells

  • Combine with MICAL3 antibody staining to correlate oxidation events with MICAL3 localization

• Target Identification:

  • Use MICAL3 antibodies to immunoprecipitate oxidized proteins following MICAL3 overexpression

  • Analyze these proteins by mass spectrometry to identify oxidation sites

How might MICAL3 antibodies contribute to understanding neurodegenerative diseases?

Given MICAL3's interactions with proteins like CRMP2, which are implicated in neurological disorders, MICAL3 antibodies may help investigate:

• The role of MICAL3 in axon guidance and neuronal development

• Potential dysregulation of MICAL3 activity in neurodegenerative conditions

• Interactions between MICAL3 and disease-associated proteins

What experimental approaches can combine MICAL3 antibodies with live cell imaging?

Emerging technologies allow for dynamic studies of MICAL3 in living cells:

• Antibody Fragment-Based Imaging:

  • Use fluorescently labeled antibody fragments (Fabs) derived from MICAL3 antibodies for live cell imaging

  • Track MICAL3 dynamics during vesicle trafficking or cell division events

• Single-molecule tracking:

  • Combine quantum dot-labeled MICAL3 antibody fragments with super-resolution microscopy

  • Map diffusion patterns and interaction kinetics of MICAL3 in real-time